Advances in Agronomy, Volume 29

ADVANCES IN AGRONOMY VOLUME 29 CONTRIBUTORS TO THIS VOLUME ESHELBRESLER K. R. CHRISTIAN C. S. COOPER R. C. DALAL ...

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ADVANCES IN

AGRONOMY VOLUME 29

CONTRIBUTORS TO THIS VOLUME

ESHELBRESLER

K. R. CHRISTIAN C. S. COOPER

R. C. DALAL J. DOBEREINER D. R. GRIFFITH GURDEVS. KHUSH T. MAEDA CARLOSA. NEYRA S. D. PARSONS C. B. RICHEY

W. R. SCOWCROFT

H. TAKENAKA B. P. WARKENTIN

ADVANCES IN

AGRONOMY Prepared under the Auspices of the AMERICAN SOCIETY OF AGRONOMY

VOLUME 29

Edited by N. C . BRADY International Rice Research Institute Manila, Philippines ADVISORY BOARD

w.L. COLVILLE, CHAIRMAN G. W. KUNZE D. G. BAKER D. E. WEIBEL G. R. DUTT H. J. GORZ M. STELLY, EX OFFICIO, ASA Headquarters 1977

ACADEMIC PRESS 9 New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT @ 1977, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY B E REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM T H E PUBLISHER.

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United Kingdom Edition prtblislied b y ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l

LIBRARY OF CONGRESS CATALOG CARD NUMBER:50-5598 ISBN 0-12-000729-0 PRINTED IN T H E UNITED STATES OF AMERICA

CONTENTS

............................ PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTRIBUTORS TO VOLUME 29

iX

xi

NITROGEN FIXATION I N GRASSES

Carlos A. Neyra and J . Dobereiner I . Introduction

.................................... ................. 111. Bacteriology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Factors Affecting Nitrogen Fixation in Grasses . . . . . . . . . . . . . . V . General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Nitrogen Fixation in C-3 and C-4 Grasses

1 3 12 26 30 33 38

SOMATIC CELL GENETICS AND PLANT IMPROVEMENT

W . R. Scowcroft I. I1. 111. IV. V. VI . VII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant Cell Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anther Culture and Haploids . . . . . . . . . . . . . . . . . . . . . . . . . Mutant Isolation and Selection . . . . . . . . . . . . . . . . . . . . . . . . Plant Cell Protoplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Transformation in F'lants ....................... Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 40 44 48 55 61 73 74

SOIL ORGANIC PHOSPHORUS R. C. Dalal I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Organic Phosphorus Content of Soil ..................... 111. Nature of Soil Organic Phosphorus ...................... IV Organic Phosphorus in Soil Solution ..................... V . Organic Phosphorus Turnover in Soil .................... VI . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

83 84 86 90 96 112 113

vi

CONTENTS

GROWTH OF THE LEGUME SEEDLING

C. S. Cooper

I. I1. I11. IV . V. VI . VII . VIII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological Predetermination . . . . . . . . . . . . . . . . . . . . . . . . Germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stages of Seedling Development . . . . . . . . . . . . . . . . . . . . . . . Improvement of Legume Seedling Vigor . . . . . . . . . . . . . . . . . . Seedbed Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seeding Forage Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seeding Management Practices . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119 120 121 123 130 130 131 137 137

YIELDS AND CULTURAL ENERGY REQUIREMENTS FOR CORN AND SOYBEANS WITH VARIOUS TILLAGE-PLANTING SYSTEMS

C . B . Richey. D . R . Griffith. and S . D . Parsons

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Tillage-Planting Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Influence of Tillage-Planting System on Yields . . . . . . . . . . . . . IV . Yield Factors Influenced by Tillage-Planting System . . . . . . . . . . V . Energy Requirements for Various Tillage-Planting Systems . . . . . Vl . Projecting Energy Savings with Reduced Tillage . . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141 143 147 157 169 178 180 180

EFFECTS OF THE ENVIRONMENT ON THE GROWTH OF ALFALFA

K . R . Christian I . Introduction

....................................

............. 111. Shoot Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Root Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Genetic Variation in Response t o Environment

V . Environmental Factors and Vegetative Growth . . . . . . . . . . . . . . VI . Phases in Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vll . Plant Associations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI11 . Genetic Adaptation t o Environment ..................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183 185 186 189 191 209 214 217 219

CONTENTS

vii

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

T . Maeda. H . Takenaka. and B . P. Warkentin I. I1. I11. IV . V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of Allophane Soils . . . . . . . . . . . . . . . . . . . . . . . . . . Physical Characteristics of Allophane Soils . . . . . . . . . . . . . . . . Soil Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

229 232 241 246 253 261

DISEASE A N D INSECT RESISTANCE IN RICE

Gurdev S. Khush I. I1. 111. IV . V. VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disease Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insect Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developing Varieties with Multiple Resistance . . . . . . . . . . . . . . Stability of Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

265 266 301 322 329 333 333

T R IC K LE-D R IP IR R IG A T I0N: PR INC IPL ES A N D APPLICATION TO SOIL-WATER MANAGEMENT

Eshel Bresler

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Potential Advantages of Trickle Irrigation . . . . . . . . . . . . . . . . . 111. Problems in Practical Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Modeling of Water and Salt Flows . . . . . . . . . . . . . . . . . . . . . . V . Soil-Water Regime during Trickle Infiltration . . . . . . . . . . . . . . . VI . Solute Distribution during Infiltration . . . . . . . . . . . . . . . . . . . VII . Application of Infiltration Models to the Design of Trickle Irrigation Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Water Management in Marginal Soils . . . . . . . . . . . . . . . . . . . . . List of Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

376 387 389 391

SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

395

344 345 351 353 367 372

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CONTR I BUT0 RS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ESHEL BRESLER (343), Division of Soil Physics, lnstitute of Soils and Water, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel K. R. CHRISTIAN (183), Division of Plant Industry, Commonwealth Scientific and Industrial Organization, Canberra, Australia C. S. COOPER (1 19), Agricultural Research Service, United States Department of Agriculture, Bozeman, Montana R. C. DALAL (83), Department of Agronomy and Soil Science, University of New England, Armidale, N. S. W., Australia J. DOBEREINER ( l ) , EMBRAPA, Campo Grande, Rio de Janeiro, Brazil D. R. GRIFFITH (14 I), Purdue Agricultural Experiment Station, Lafayette, Indiana GURDEV S . KHUSH (265), International Rice Research Institute, Los BaCos, Philippines T . MAEDA (229), Department of Agricultural Engineering, Hokkaido University, Sapporo, Japan CARLOS A. NEYRA (l), EMBRAPA, Campo Grande, Ria de Janeiro, Brazil S. D. PARSONS (141), Purdue Agricultural Experiment Station, Lafayette, Indiana C. B. RICHEY (141), Purdue Agn’cultural Experiment Station, Lafayette, Indiana W. R. SCOWCROFT (39), Division of Plant Zndusty, Commonwealth Scientific and Industrial Organization, Canberra, Australia H. TAKENAKA (229), Department of Agricultural Engineering, University of Tokyo, Tokyo, Japan B. P. WARKENTIN (229), Department of Renewable Resources, McGill University, Montreal, Canada

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PREFACE The internationality of science has never been more evident than it is today. And the need for international exchange of scientific findings is as pressing in soil and crop science as in any other field. Meeting the world's food needs while simultaneously maintaining an environment suitable for humankind as well as other animal species is among the primary objectives of soil and crop scientists throughout the world. These objectives can be attained only if there is rapid and effective interchange of scientific information from one country to another. This volume follows the pattern that has characterized recent issues of Advances in Agronomy. The subjects addressed are of international concern. Likewise, the authors are from diverse backgrounds and nationalities. While all of the papers in this volume provide information of concern to those who produce food, four have rather direct implications for food production. The review of research on nitrogen fixation on grasses, on the pests of rice, on trickle-drip irrigation, and on somatic cell techniques for plant improvement each identify unique means of increasing world food supplies. The authors have appropriately emphasized past accomplishments, current research constraints, and potential for future progress. Two papers deal with soil characteristics. One provides an upto-date review of soil organic phosphorus research. The other is concerned with allophane, an important soil mineral, the knowledge of which has been quite limited. In a third soils-oriented paper, energy-saving minimum tillage techniques are discussed. This is a research area that will receive increasing attention in the future because of rising energy costs. Research on forage crops is the focus of two papers, one concerned with the growth of legume seedlings and the second with the influence of the environment on alfalfa growth. Both reviews will be helpful for animal-oriented food production systems. Soil and crop scientists throughout the world are indebted to the 14 authors who have prepared these important papers. Their contributions will undoubtedly stimulate future research efforts in the subject areas covered in this volume. N. C. BRADY

xi

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NITROGEN FIXATION I N GRASSES Carlos A. Neyra and J. Dobereiner EMBRAPA, Campo Grande, Rio de Janeiro, Brazil

I. Introduction .................................................. 11. Nitrogen Fixation in C-3 and C-4 Grasses . . . . . . . . . . . . . . . .. . . . . . . . . . A. C-4 Grass Systems . . . . . . . . . . . . . . . . . . ~. . . . . ~. . . . . . . . . . . . . B. C-3 Grass Systems . , . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... .. . . . . . . . . . .. . . ... C. Miscellaneous Systems . . . . . . . ..... . . 111. Bacteriology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Beijerinckia . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . .. . . .. . . . . B. Azotobacter paspali . C. Spirillum lipoferum . . .. . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . IV. Factors Affecting Nitrogen Fixation in Grasses . . . . . . . . . . . . . . . . . . . . . . A. Seasonal and Diurnal Fluctuations . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. PlantGenotype .............................................. . .. .. . . . . .. .. .. . . . . . . .. .. . . . . . . . . .. . . . . C. Temperature . . D. Oxygen .................................................... . . .. . . . . . . . . . . . . . . .. . . . . . . . E. Combined Nitrogen . . . . . . . . V. General Discussion . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .................................................... Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 3 5 9 11 12 13 14 15 26 26 27 28 28 29 30 33 38

I. Introduction

Population growth and changing dietary habits have led to an increased demand for protein for human consumption. Combined nitrogen (N) and biological Nzfutation represent the major inputs of N for crop and protein yield. While some plants, notably grain crops, have relied mostly on combined N sources, some other plants, notably legumes, have the capability of being at least partially self-sufficient through symbiotic Nz fixation. As to the nonbiological inputs, increased use of fertilizer N is probably the most important single factor that has enabled cereal grain production to increase significantly in recent years (Hardy, 1976). It is also predicted that increasing cereal grain production at the world level will require the use of increasing amounts of fertilizer N (Hardy, 1976). However, in the less developed countries the availability and the high prices of fertilizer N are limiting factors for its use on a large scale. In addition, in tropical regions considerable amounts of N, mostly in the form of NO3 are lost from the soil by leaching (da Eira et al., 1968). 1

TABLE I

N, Fixation and Incorporation in Digitaria decurnbens and Paspalurn notaturn Grown in Pots for 2 Weeksa N, fixed

Plant species and amendment Digifaria decurnbens cv. transvala without sucrose*

D. decumbens cv. slenderstem with 0.5% sucrose

Paspalurn notaturn cv. batatais without sucroseb

P. notaturn cv. batatis with 0.5% sucrose

Atoms % excess l 5 N

Part of plant

Total N in plants (mglpot)

Roots Rhizomes Stems Leaves Total

1.78 2.74 4.32 7.34 16.18

0.151 0.146 0.021 0.007

Roots Rhizomes Stems Leaves Total

1.40 4.06 1.80 4.74 12.00

0.582 0.709 0.073 0.010

Roots Rhizomes Leaves Total

2.89 2.62 6.03

0.563 0.703 0.070

11.54

Roots Rhizomes Leaves Total

2.71 3.25 3.85 9.81

-

-

-

1.021 1.392 0.053 -

(fig/pot)

2.69 4.00 0.91 0.5 1 8.1 1 8.15 28.79 1.31 0.47 38.72

16.34 15.28 2.97 34.59 27.56 44.39 2.06 74.01

( d g roots + rhizomes) -

-

10.73 -

-

33.64 -

24.39 -

43.33

'Summarized from De-Polli et al. (1977). See Soil Biol. Biochern. 9, 119-1 23. Used by permission. 'Values from D. decumbens are from single pots but those from P. nofafumare from duplicate pots. All pots were incubated in the same jar for 72 hours (15-hOur light and 9-hour dark periods) in a gas mixture containing an average 42.8% N, (enrichment I5N, 85.5%), 2.6% O , , 3.2% CO,, and 51.4% A. The cv. for the mass spectrometer analyses was 0.40-2.62%.

NITROGEN FIXATION 1N GRASSES

3

Although improved technologies of fertilizer N production and increased efficiency of fertilizer use by plants could make more N available for the plants, nevertheless alternative technologies should be found to lessen the dependence of plants on fertilizer N. To develop N self-sufficiency in forage grasses and grain crops may constitute a major breakthrough in the years ahead. Efforts along these lines may include the incorporation of nifgenes into cells that normally do not fur N2 (Brill, 1974) or the development of already present plant-bacteria associations. Dixon and Postgate (1972) demonstrated the possibility of transferring nifgenes to bacteria; Giles and Whitehead (1976) have demonstrated that Nz -fixing bacteria can be incorporated directly into protoplasts of a mycorrhizal fungus. This could be of tremendous importance in the infection process of plant roots by N2 -fixing bacteria. Recent findings (Rinaudo et al., 1971; Dobereiner et al., 1972a; Dobereiner and Day, 1976; von Bulow and Dobereiner, 1975) have revealed already existing associations of tropical grasses with N2 -fixing bacteria which under favorable conditions may be contributing significantly to the nitrogen economy of these plants. Although it is premature to predict the actual contribution of Nz fixation to plant nitrogen, it is of major importance to mention that incorporation of ''N2 into plant tissues has recently been demonstrated (Table I). Many of the tropical grasses able to support significant nitrogenase activity possess the photosynthetic C-4 pathway (Day et al., 1975b). The amount of light required to saturate photosynthesis and the maximum photosynthetic rate attainable are much greater in C-4 than in C-3 plants (Chollet and Ogren, 1975). At high light intensities and low temperatures the rate of photosynthesis is essentially the same in C-3 and C 4 species, but at higher temperatures C 4 plants show higher photosynthetic rates. Furthermore, losses of carbon due to photorespiration are minimal in C-4 plants (Chollet and Ogren, 1975). This evidence suggests that tropical grasses may be very efficient in harvesting light energy for nitrogen fixation. Maximization of N2 futation in tropical grass-bacteria associations and the elaboration of agronomic practices to enhance or promote N2 fixation in grasses will depend on the identification of the various limiting factors controlling this process under field conditions. In this review we intend to give an interpretative account of recent developments in this rapidly expanding field and to discuss in more detail some of our work which is not yet available in the literature.

II. Nitrogen Fixation in C-3and C-4 Grasses

Nitrogen-futing bacteria are widely distributed in soils, and it has been suggested for a long time that major contributions to the system could be expected

4

CARLOS A . NEYRA AND J. DOBEREINER

(Beijerinck, 1925; Schroder, 1932; Krasil'nikov, 1968; Dobereiner, 1966; AbdEl-Malek, 1971; and many others). Various anaerobic, facultative, and aerobic bacteria are capable of fixing nitrogen in soil, in the rhizosphere, and in roots. Interest in aerobic organisms has been generally greater because aerobic metabolism is more efficient, and because agricultural soils, with some exceptions (e.g., paddy rice), are well aerated. In most systems the availabitity of energy and carbon substrates represents the major limiting factors to biological N2 fixation. Nitrogen fixation of importance in soils has only been demonstrated after the addition of carbon substrates (Mishustin, 1970; Brouzes et al., 1971; Abd-ElMalek, 1971) or when growing plants release part of their photosynthates (Dobereiner and Alvahydo, 1959; Dobereiner, 1961; Dobereiner et ul., 1972a; Day et ul, 1975b). In addition, plant root exudates can play an important role in the establishment and maintenance of the rhizosphere population (Rovira, 1965b). A good example of solar energy utilization for N2 fixation is the legume symbiosis, where the energy requirement for nitrogen fixation is equivalent to the requirement for nitrate reduction (Minchin and Pate, 1973; Gibson, 1976). However, photosynthate availability is still considered a major limiting factor for N2 fixation in soybeans (Quebedeaux et ul., 1975). On the other hand some tropical grasses can grow and produce constant yields without addition of nitrogen fertilizer to the soils, and it was suspected for many years that substantial Nz fixation occurred in these systems (Parker, 1957; Moore, 1966; Dobereiner, 1966). Because of their photosynthetic characteristics (see Section I), most of these plants are in a favorable position with regard to photosynthate availability for growth and N2 fixation. In the last 5 years the evidence for Nz fixation in grasses has accumulated rapidly. The results obtained by several authors for field-grown tropical plants are summarized in Table 11. Although the measurement of uptake of "N-enriched N z represents the most satisfactory method for evaluation of N2 fixation, the introduction of the acetylene reduction method has represented a major breakthrough in the evaluation of N, fixation both in the laboratory and under field conditions. The work of Schollhorn and Burris (1967) and Dillworth (1966) suggested that the rate of acetylene reduction may be used as an index of the rate of Nz fixation. The reduction of acetylene to ethylene (C, H2-C2 H4) and the measurement of ethylene by gas chromatography has been extensively used for the assessment of N2 fixation in grass-bacteria associations. The reader is referred to the literature for further details on the use of "N as a tracer and for the acetylene reduction method (Burris and Wilson, 1957; Stewart et uL, 1967; Hardy et al., 1968, 1973; Burris, 1972, 1974; Dart et ul., 1972). Several procedures for the assessment of N2 fixation by acetylene reduction have been adopted. A general procedure for assaying excised roots was described

NITROGEN FIXATION IN GRASSES

5

for Paspalurn notaturn (Dobereiner et al., 1972a) and Dig'taria decurnbens (Abrantes et al., 1975), and the same procedure with slight modifications has been used for several other forage grasses and grain crops (von Biilow and Dobereiner, 1975; Day et al., 1975b; van Berkum and Neyra, 1976; Sloger and Owens, 1976). Steel cylinders of small diameter are very useful for taking cores of small grasses from the field (Day et al., 1975a; Abrantes et al., 1975). A variety of devices have also been described for in situ measurements of N2 fixation under field conditions (Balandreau et al., 1974; Balandreau, 1975; Watanabe and Kuk-Ki-Lee, 1975). A. C-4 GRASS SYSTEMS

I . Paspalurn notatum The first tropical C-4 grass-bacterial association to be studied in detail was that of Paspalurn notaturn-Azotobacter paspali. Five ecotypes in this grass (tetraploid types) show a very specific association with Azotobacter paspali (Dobereiner, 1966; Dobereiner and Campelo, 1971). Of the 33 ecotypes or cultivars studied, only five (tetraploid types) stimulated A. paspali growth in the rhzosphere. Establishment of the bacteria on the roots takes several months and inoculation does not accelerate this rhizosphere association (Dobereiner and Campelo, 1971). Field plants, transplanted with adhering soil into vermiculite and watered with nitrogen-free nutrient solution, fixed 80 mg N per pot in 2 months, the amount necessary for normal growth (Dobereiner and Day, 1975). CzH2 reduction assays with intact soil plant cores correlated well with excised roots extracted from the soil and assayed after overnight preincubation in low p 0 2 (Dobereiner et al., 1972a). Paspalurn notatum grown in sand, from seeds, did not show A. paspafi establishment, except when glucose was added (Kass et al., 1971). It is possible that besides A. paspali other microorganisms (e.g., mycorrhizal fungi) may be involved in the establishment of the association. Mosse (1972) observed very intensive mycorrhizal infection of this grass. Inoculation of irradiated Brazilian soils with Endogone spores resulted in large increases in forage yield in Paspalurn notaturn (Mosse, 1972). Localization of A. paspali has been suggested to be in the mucagel layer outside the root (Dobereiner et al., 1972a). The correlation of root piece nitrogenase activity and enrichment culture activity in A . paspali sucrose medium was highly significant (r = .08l) (Dobereiner and Day, 1974) when the same root pieces were used for inoculation of the enrichment medium. Estimates of nitrogen fixation in intact soil plant cores (10 cm #) by the CzH2 reduction method were calculated to be 340 g N/ha per day. "N2 assays in smaller vessels extrapolated to 110 g N/ha per day (calculated from data by De-Polli, 1976).

6

CARLOS A. NEYRA AND J. DOBEREINER

2. Sugar Cane In many parts of the world this crop has been grown in monoculture for more than 100 years without addition of nitrogen fertilizer and a survey in SBo Paulo (Brazil) revealed that only half of the fields with this crop responded to nitrogen fertilizer even if PK was also supplied (Verdade, 1967). Selective stimulation of the nitrogen-fixing Beijerinckia under sugar cane vegetation and positive rhizosphere effects have been shown (Dobereiner, 196 1). Assays with the acetylene reduction method indicate that in this crop only a minor part of the N2 is fixed in or on the roots and most of it in the rhizosphere or in the soil (Dobereiner et al., 1972b; Ruschel, 1976). Rain water can carry leaf exudates into the soil which enhance Beijerinckia growth (Dobereiner and Alvahydo, 1959). Maximal soil nitrogenase activities were found in rhizosphere soil and between the rows, where the canopy closes (Dobereiner et al., 1972b). Sugar cane seedlings exposed to 's N2 indicated fixation, incorporation, and translocation of nitrogen to the leaves (Ruschel et al., 1976). Spirillum lipofem8mdoes not appear to be stimulated in the sugar cane rhizosphere (Dobereiner, 1976a) and this supports the prevalence of Beijerinckia spp. as the major N2 fixer in this plant.

3. Digitaria decumbens This grass contains several cultivars of agronomic importance, e.g., Pangola, Transvala, and Slenderstem. These three grasses were grown in our experimental fields from November 1973 to May 1975 (two summers, one winter) and showed mean nitrogen yields of 1S O , 1.48, and 1.40 kg/ha per day, respectively. The nitrogen gain of the soil (0-20 cm depth) calculated from Kjeldahl analyses before and after this period was 405, 216, and 468 g/ha per day, respectively (Schank et al., 1975). Intact soil plant core assays in the summer 1975 showed nitrogenase activities equivalent to 880, 480, and 970 g N/ha per day, respectively (Day and Dobereiner, unpublished data). Similar values (1460 ? 85 g N/ha per day for Transvala and 1326 g N/ha per day for Slenderstem) have been estimated from the data of De-Polli (1976). In lsN2 experiments significant incorporation and translocation was shown in both species (Table I). The N2 -fixing bacteria most commonly associated with Digitaria decumbens is Spirrillum lipofemm. In several experiments, significant correlations of root piece nitrogenase activity with S. lipofemm enrichment culture activity were found, suggesting that S. lipofemm is the major organism responsible for nitrogenase activity on roots (Dobereiner and Day, 1976). The most active root pieces showed strongly reducing sites within the cortex, where cells packed with tetrazolium-reducing bacteria were found. Inactive root pieces did not show such sites (Dobereiner and Day, 1976).

TABLE I1 Potential of N, Fixation in Field-Grown Tropical Forage Grasses Associated with N, -Fixing Bacteria

N, -ase activity C, H, /h/g Plant species Andropogen gayanus (C, ) Aizdropogen spp. (C,) Brachiaria mutica (C,) B. rugulosa (C,) B. brachylopha (C,) Bulbostylis aphylanthoides Cynoden dactilon (C,) Cynoden dactilon (C, ) Cyperus rotundus (C, ) Cypents sp. (?) Cyperus obtusiflorus (?) Digitaria decumbens (C,) Hyparrhenia rufa (C,) Hyparrhenia rufa (C,) Hyparrhenia dissoluta (?) Melinis minutiflora (C,) Panicum maximum (C, j Panicum maximum (C,) Paspalum notatum (C,) Paspalum comersenii (?) Pennisentm purpureum (C, 1 Pennisetum purpureum ( C , )

Country

Roots

Soil

References

Nigeria Ivory Coast Brazil Brazil Ivory Coast Ivory Coast Brazil Nigeria Brazil Nigeria Ivory Coast Brazil Brazil Nigeria Ivory Coast Brazil Brazil Nigeria Brazil Nigeria Brazil Nigeria

15-270 50-380 150-750 5-150 100-140 74 11-269 10- 50

-

Day and Dart (personal communication) Balandreau e t al. (1973) Dobereiner and Day (1975) Dobereiner and Day (1975) Balandreau et al. (1973) Balandreau et al. (1973) nobereiner and Day (1975) Day and Dart (personal communication) Dobereiner et al. (1975) Day and Dart (personal communication) Balandreauet al. (1973) Dobereiner and Day (1975) Dobereiner and Day (1975) Day and Dart (personal communication) Balandreau e t al. (1 973) Dobereiner and Day (1975) Dobereiner and Day (1975) Day and Dark (personal communication) Dobereiner and Day (1975) Day and Dart (personal communication) Dobereiner and Day (1975) Day and Dart (personal communication)

-

0 -

0-0.07 -

10- 30

-

2 30-620 21-404 20- 30 30-140 10- 15 13- 41 20-299 75 2-283 25-30 5-954 60

-

nMinimal and maximal values obtained with excised preincubated roots.

-

0-0.35 0-0.15 -

04.19 04.15 -

04.33 -

04.09 -

8

CARLOS A. NEYRA AND J. DOBEREINER

4. Other Forage Grasses Excised root assays have shown that several other tropical C-4 forage grasses are able to fur N2 (Table 11). In a 3-year experiment in Nigeria, soil under fallow of Panicum maximum contained 0.18% N (15 cm depth) while fallows under legumes (Leucena glauca and Cajanus cajan) contained only 0.13%. This difference corresponds to 250 kg N/ha per year (Greenland, 1975). This illustrates the tremendous importance of such a fallow crop for the nitrogen balance of tropical soils even if only part of this amount was due to bic Jgical N2 fixation and the remaining to prevention from leaching or denitrification. Only limited results from core assays are available. Balandreau and Villemin (1973) estimated N2 fnation (C2 H,) rates of 10-1 5 kg N/ha per year (in situ assays) in Ivory Coast savannas where Panicum maximum and Andropogon sp. were predominant. These authors found N,-fixing aerobes to be predominant in the rhizosphere but did not identify them or relate them specifically to root nitrogenase activity. A survey of S. lipoferum occurrence in various parts of Brazil revealed a high incidence of this organism where Panicum maximum replaced virgin forest (Diibereiner et al., 1976). In another experiment (six sites, 10 samples each) there was a significant difference in S. lipoferum incidence between forage grasses. Panicum maximum and Brachiaria mutica were the most favorable and Hypawhenia rufa the least (Dobereiner, 1976b). The mode of infection of Panicum maximum by Spirillum lipoferum has been investigated by electron microscopy in axenic seedlings (Garcia et al., 1976). The bacteria were observed on the root surface within 24 hours and in the middle lamellae of the root cells within a week. No intracellular infection was observed even after 1 month. These authors have suggested that S. lipoferum enters the roots with the aid of pectolytic enzymes. 5. Grain Crops

Maize and sorghum represent two of the major grain crops in the world. High nitrogenase activities (up to 9000 nmoles CzH4/g roots per hour) were found on excised, preincubated maize and sorghum roots in a lowland soil in Rio de Janeiro State (von Bulow and Dobereiner, 1975). Other estimates by this method range between 100 and 2000 nmoles CzH4/g roots per hour (von Bulow and Dobereiner, 1975; Abrantes et nl., 1976; Barber et al., 1976; Okon et al., 1977a; Sloger and Owens, 1976). However, very low or no activities were reported from soil plant core and in situ assays (Balandreau and Dommergues, 1973; Barber et a l , 1976; Burris, 1976; Tjepkema and van Berkum, personal communication; for discussion on this discrepancy see Section V). In Rio de Janeiro (von Bulow and Dobereiner, 1975), Brasilia and Londrina (Peres, Nery, and Dobereiner, unpublished data) Spirillum lipoferum was found

NITROGEN FIXATION IN GRASSES

9

to be abundant in all Nz-fixing maize and sorghum roots examined. Sloger and Owens (1976) also report isolation of this organism from maize roots grown in Beltsville, Maryland, while it was not found in Wisconsin or Oregon (Burris et al., 1976; Barber et al., 1976). Field-grown maize plants in Wisconsin inoculated with strains of S. lipofemm isolated from Digitaria roots in Brazil, showed establishment of the bacteria inside the roots (Burris, 1976; Dobereiner et al., 1976). Inoculated plants showed higher nitrogenase activity than uninoculated ones, while nitrogen-fertilized plants had no activity (Burris et al., 1976; Barber et al., 1976). The total number of bacteria in surface-sterilized maize roots was similar t o the number of S. lipofemm in the inoculated maize roots (Okon et al., 1976a). Significant correlations (p = 0.01) between maize root piece activities and enrichment culture activities were only obtained when the roots were previously surface-sterilized (von Bulow and Db'bereiner, 1975). Detailed studies on the localization of Spirillum lipofemm in maize and sorghum roots are not yet available. Maize plants grown in sterilized sand and soil collected in Wisconsin, showed nitrogenase activities when inoculated with S. lipofemm. The organism was reisolated from surface-sterilized roots (Burris et al., 1976). Effects on plant growth and nitrogen incorporation however were not significant in these experiments. In Oregon attempts to isolate Nz-furing bacteria from maize plants yielded Enterobacter cloacae (Raju et al., 1972).

B. C-3 GRASS SYSTEMS 1. Rice

There is little doubt as to the substantial contribution of biological Nz fixation to the N economy of this most important grain crop. For instance, a total of 23 rice crops, in an 11-year experiment at the International Rice Research Institute in the Philippines, were obtained from a nonfertilized field with no apparent decline in the nitrogen fertility of the soil. About 45 to 60 kg N/ha per crop were removed through straw and grain (Watanabe and Kuk-KiLee, 1975). This represents a substantial amount of N which had to be replaced in order to maintain the fertility level of the soil. Blue-green algae and photosynthetic bacteria account for a large part of the Nz fixation in paddy rice (Watanabe and Kuk-Ki-Lee, 1975; Elnawamy, 1976). This subject has been reviewed elsewhere (Stewart, 1976; Venkataraman, 1975). Bacterial Nz f i a t i o n in intact rice cultures grown in test tubes has been shown by Rinaudo et al. (1971) by Kjeldahl analyses and acetylene reduction. For the latter method, plants were removed and assayed after 24 hours of preincubation under anaerobic conditions. The results obtained by the two methods were in

10

CARLOS A. NEYRA AND J . DOBEREINER

good agreement. Excised root assays of field-grown rice roots (Yoshida, 1971a; Yoshida and Ancajas, 1973) confirmed “rhizosphere N2 fixation.” Results from intact soil plant systems in the field gave about 50 to 200 g N/ha per day at the flowering stage, by the nonalgal component. The algae were separated by removing the flooding water and assayed separately for N2 fixation (Watanabe, 1976). Balandreau (1975) reported that 25 to 30 kg N/ha can be fixed for the growing season by the nonalgal component. Bacterial counts indicate that Beijerinckia sp. and Enterobacter cloacae are the most common N2-furing bacteria in the rhizosphere of rice (Yoshida, 1971b; Balandreau, 1975). However, the methods used by these authors would not reveal Spirillum lipoferum. When various types of roots were compared, mature roots with many laterals were the most active ones (Hamad-Fares et aZ., 1976). Such roots, surface-sterilized for 1 hour with 1% chloramin T yielded almost pure cultures of an organism with properties resembling S. lipoferum (Diem et al., 1976). However, most of the nitrogen fixation in the rice system has been attributed to rhizosphere soil rather than roots themselves (Yoshida and Ancajas, 1973). Higher numbers of aerobic than of anaerobic N2-fixing bacteria in the rhizosphere of rice were also found by Dommergues et al. (1973) and Watanabe and Kuk-Ki-Lee (1975). Aerobic or microaerophilic N2-fixing bacteria were also found to be prevalent in roots of a salt marsh grass (Patriquin, 1976). Methaneoxidizing bacteria which are able to fix Nz were also found in rice paddies. The large amount of CH4 which can accumulate in these soils, should not be overlooked as a potential carbon source for Nz fixation (De Bont et aZ., 1976). However, O2 diffusion seems a limiting factor for this system. Inhibition of CH4 oxidation by C2H2 and consequent interference in C2 H2 reduction complicate estimates of Nz fixation where these organisms are present (De Bont and Mulder, 1976). Very high numbers (up to 3.6 X lo7) of N2-fixingCH4-oxidizing organisms were found in the rice rhizosphere (De Bont el al., 1976).

2. Wheal A nitrogen balance study in the famous Broadbalk continuous wheat experiment carried out from 1843 to 1967 in England, showed an average annual gain of 34 kg N/ha, of which 24 kg N/ha were removed with straw and grain (Jenkinson, 1973). However, values extrapolated from C2H2 reduction assays on cores were much lower (2 to 3 kg N/ha per year) (Day et al., 1975a). It was also shown that nitrogenase activity of soil cores containing wheat was significantly higher than in bare soil (Day et al., 1975a). Wheat cores assayed in Oregon have been calculated to fm 2 g N/ha per day (Barger et al., 1976). Much higher nitrogenase activities have been observed in wheat cores assayed in Rio de Janeiro (Table 111). Similar results were obtained with cores from several wheat cultivars grown in pots in Parana (Brazil) (New and Abrantes, personal communication). Excised root assays underestimated, by

11

NITROGEN FIXATION IN GRASSES

TABLE 111 Nitrogenase Activity in 10 Intact Wheat Cores (cv. Sonora) Collected at Random in the Field, at Flowering Stage

Mean 2 most active cores Mean 5 intermediate cores Mean 3 least active cores Mean all cores

nmol C, H, /hour/core

g N, /day 10 cm@core

2641 1137 180

597 x 276 X 44 x 10-6

-

-

g N, /daylhaa

506 238 38 229

aEstimate by the theoretical C, H, :N, 3: 1 ratio from 24-hour rates based on the @ of 10-cm area of the cores corrected for 15-cm distance between rows.

about one-half, the core activities but showed significant correlations with the core assays (r = 0.86 in Rio de Janeiro and r = 0.87 in Parana). In the Broadbalk experiment a large part of Nz fixation was attributed to blue-green algae but root nitrogenase activity was attributed t o anaerobic or facultative bacteria (Day et al., 1975a). Barber el al. (1976) isolated N,,-fixing strains of Enterobacter cloacae, Bacillus macerans, and B. polymyxa from wheat roots in Oregon. On the other hand, enrichment cultures in semisolid N-free malate medium inoculated with surface-sterilized wheat roots obtained from different locations in Brazil (Rio de Janeiro, Parana, and Brasilia) yielded almost 100% positive samples of SpiriZlum lipofemm. Samples from R o Grande d o Sul (extreme south of Brazil) showed that only 20% of the root samples were positive for this organism. Attempts to correlate root piece nitrogenase activity with enrichment culture activity in wheat have been unsuccessful. Larson and Neal (1976) described a highly specific association of a facultative Bacillus sp. with a disomic chromosome substitution line of wheat. The Bacillus was isolated from a soil where wheat had been growing for 30 years without nitrogen fertilizer. The rhlzosphere of this wheat line contained also more nitrate-reducing bacteria and a lower total number of microorganisms. In monoxenic culture, the bacterium closely associated itself with the root surface. Abundant numbers of bacterial cells were found on the root surface as well as in the intercellular spaces between the cortical root cells. Rovira (1965a) reported establishment of an N,-fixing Bacillus sp. in wheat. Recent fine structure studies by Foster and Rovira (1976) showed active penetration of wheat cortex cell walls by bacteria, including Bacillus sp., at the flowering stage.

C. MISCELLANEOUS SYSTEMS

In addition to the N, -Axing systems previously described, which all bear some relation to agricultural crops, a number of water plants and weeds have been

12

CARLOS A. NEYRA AND J. DOBEREINER

shown to exhibit substantial nitrogenase activity. An understanding of these systems may help to clarify others of more immediate agricultural importance. The tropical marine angiosperms K5alassia testudinum, Syringodium Jiliforme, and Diplanthera wrightii and the temperate Zostera manna fixed an amount of nitrogen sufficient for growth (Patriquin and Knowles, 1972). Thalassia testudinum Nz furation reached 100 to 500 kg N/ha per year. Conversion factors of CzH2 reduction estimates as compared with estimates by lsNz incorporation were close to the theoretical value 3 (2.6 to 4.6). A good correlation of numbers of anaerobic Nz -furing bacteria and nitrogenase activity in glucose-amended sediments was obtained, but aerobic N2 fixers were 50 to 300 times more abundant in the rhizosphere than in the sediment and the authors concluded that organisms other than Azotobacter and Clostridium are the predominant nitrogen furers in these systems. Spartina altemiflora a C-4 grass from Canadian salt marshes (Patriquin, 1976) was shown to have an association similar to that described for Digifaria (Dobereiner and Day, 1976). Tetrazolium-reducing bacteria, similar to S. lipofemm, were found to be concentrated in the outer and inner cortex layer of the roots. Nz-fixing aerobic bacteria resembling S. lipoferum were also isolated from Potamogeton Jilifonnis roots grown in Scottish lakes (Silvester-Bradley, personal communication). Kgh nitrogenase activity has alwo been observed in excised roots of mangroves (Rhizophora mangle and two other species; Silver et al., 1976) and in intact soil plant cores of Juncus balticus (Barber et al., 1976) and several inulin-containing plants (Dahlia pinnata and others; Jain and Vlassak, 1975; Vlassak and Jain, 1976). I I I . Bacteriology

Nitrogen-fixing bacteria which have been found in association with grasses are all capable of fixing nitrogen in soil or culture medium without the plant. Therefore, they are generally included in the group of “free-living Nz-fixing bacteria” (Mulder and Brotonegoro, 1974). An excellent up-to-date review on the entire group has been given by these authors and we will therefore restrict this chapter to bacteria for which specific associations with tropical grasses have been shown. Several species of Nz-fixing bacteria have been isolated from the rhizosphere of temperate plants, e.g., the facultative Enterobacter cloacae, other Enterobacter spp., and members of the Klebsiella aerobacter group (Raju et al., 1972; Evans et al., 1972; Barber et al., 1976; Balandreau, 1975) but have not been shown to be involved in nitrogenase activity on roots or in the rhizosphere. A microaerophilic N2-fixing bacterium has been isolated from Digitmia sanguinalis with characteristics very similar to s. lipoferum (Barber and Evans, 1976). The Azotobacter spp. (except A. paspali) are found mainly in the outer rhizosphere of plants and can be very abundant under warm arid conditions

NITROGEN FIXATION IN GRASSES

13

(Vancura et al., 1965; Abd-El-Malek, 1971). Rice in Japan and India (Ishizawa and Toyoda, 1964; Gopalakrishnamurthy et al., 1967) and sorghum in India (Shantaram and Rangaswamy, 1967) have been shown to stimulate Azotobacter growth. Books have been written on the presence of Azotobacter in the rhizosphere of plants (Rubenchik, 1963; Krasil’nikov, 1958) but, in more recent reviews, Macura (1966), Mishustin (1970), and Rovira (1963) have come to the overall conclusion that the growth of Azotobacter chroococcum and A. vinelandii is not influenced by plant roots. Even inoculation does not normally establish these bacteria in the rhizosphere (Dobereiner, 1974). In some instances establishment of Azotobacter on the roots has been observed, particularly when the competitive rhizosphere microflora was eliminated (Riviere, 1959; Jackson and Brown, 1966; for a more detailed discussion on this subject refer to Diibereiner, 1974). Inoculation of Ammophyla arenaria (a sand grass) with Azotobacter chroococcum had beneficial effects on plant growth and nitrogen content (Abdel Wahab and Wareing, 1976). When rice seedlings in axenic cultures were inoculated with Azotobacter chroococcum and two other bacterial isolates from rice, acetylene reduction was observed. However, there was no effect on plant growth. Even the highest activity observed (0.2 pg N per plant per day) was too low t o meet the need for nitrogen (400 pg N in 3 weeks) (Watanabe, 1975). Inoculation of rice seedlings (grown in nonsterile soil) with Azotobacter or Beijerinckia in France gave no increase in nitrogenase activity (MourarrCt et al., 1975). However, inoculation with Spirillum lipoferum resulted in a threefold increase in nitrogenase activity (Mourarrbt et al., 1975). Anaerobic N2 -fixing bacteria are seldom stimulated in the rhizosphere. Still, Katznelson ( 1 965) found that Clostridium numbers were increased in the rhizosphere of some plants and Rovira (1963) found that this organism could be established in the root zone of wheat, maize, tomato, and lucerne. The overall effect of a particular plant cover on the occurrence of certain bacteria is good evidence for meaningful rhizosphere or root associations. Currently, information on such effects is available for only three tropical N2-fixing bacteria. These will be discussed in more detail.

A. Beijerinckia

Beijerinckia species are restricted to tropical and subtropical regions (Derx, 1953; Becking, 1961; Dobereiner, 1968). The acid tolerance of these organisms enables them to compete successfully with most other soil microorganisms in acid soils but they are unable to compete in neutral soils (Strijdom, 1967). In sterile soils changes of pH had no effect. A survey of Beijerinckia distribution in several Brazilian states demonstrated the presence of this organism in 97% of the soil samples under sugar cane and

14

CARLOS A. NEY RA AND J. DOBEREINER

only 60% of the soil samples from other vegetations (Dobereiner, 1961). After transplanting sugar cane to a new field, Beijerinckia numbers in the rhizosphere and root surface soil increased steadily, but numbers of total bacteria, actinomyces, and fungi decreased. This suggests that changes in the microbial equilibrium of the sugar cane rhizosphere favor N2 -fixing Beijeiinckia. Morphological and taxonomic characteristics of Beijerinckia species together with methods for isolation and culturing have been described by several authors (Becking, 1961; Dobereiner and Ruschel, 1958, 1964; Bergey, 1975). However, very little is known about the physiology of this organism. There is no report on the in vitro preparation of nitrogenase. Nitrogenase activity assayed in vivo requires high levels of CzHz (40-80%) for saturation of the enzyme (Spiff and Odu, 1973; Dobereiner, 1973). This organism is surrounded by a tough gum which may act as a physical barrier to oxygen (or CzHz) and therefore function as an oxygen protection mechanism for the nitrogenase, as suggested by Hill (1971) for Derxiagummosa. Mutants of Beijerinckia indica with less gum seem to be more sensitive t o oxygen (Dobereiner, 1973). Very fast growth of Beijeiinckia can be obtained in liquid media aerated with an N2 :Oz :COz (95:4.5:0.5) gas mixture but little growth occurs without COz . Beijerinckia fluminensis does not form a gum but shows characteristic zooglea-like clusters of four to eight organisms, surrounded by a distinctive membrane. The function of this membrane is not known but it has been suggested that it may retard oxygen diffusion and thereby protect the nitrogenase (Dobereiner, 1974). The latter was not confirmed in chemostat cultures with known p 0 2 . Peiia and Dobereiner (1974) studied the effects of NO3- and NH4' on growth and nitrogenase activity of Beijerinckia spp. Beijerinckia indica maintained about 30% of the original nitrogenase activity for 60 hours after the addition of 10 mM N03- and 10-20% of the activity after the addition of 10 m M NH4'. In contrast, Azotobacter vineIandii lost all nitrogenase activity in 3 to 3 hours after similar additions of NO3- and NH4' (Peiia and Dobereiner, 1974). Beijerinckia fluminensis appeared to be unaffected by NO3- during a 6-hour incubation period. Even after 3 days 50% of the nitrogenase activity was still present. The capacity to fix NZ even in the presence of hgh concentrations of mineral nitrogen may be of considerable ecological importance. These bacteria have not been examined for a dissimilatory nitrate reductase.

B. A z o t o b a c t e r paspali

Azotobacter paspali, although capable of fxing N2 independently of the plant, is ecologically restricted to the rhizosphere of a few ecotypes of Pasparum notatum. This aerobic bacterium was found t o occur in 98% of the 252 root

NITROGEN FIXATION 1N GRASSES

15

surface soil samples collected in various places in Brazil and the United States. Only 3% of the soil samples from other Paspalum cultivars and 13%of the soil samples from other Paspalum species contained the bacterium (Dobereiner, 1970). Azotobacter paspali was not isolated from other plants or from soil without Paspalum. No other soil bacteria except pathogens show such specificity. This would suggest that a very close interrelationship exists between Paspalum and Azotobacter paspali. When Paspalurn notatum cuttings are transferred to new soil, there is an initial decline ofA. paspali counts on the root surface but after 4-12 months high populations are reestablished (Dobereiner and Campelo, 1971). This delay in establishment also occurs when the plants are inoculated with A. paspali. This could explain why pot experiments carried out in England were unsuccessful in establishing A. paspali on the roots of several plants (Barea and Brown, 1974; Brown, 1976). An excellent description of the properties of this organism can be found in Bergey’s Manual (1975). Although the organism has a very narrow pH range (6.0-7.0) for growth in culture media (Machado and Dobereiner, 1969), it does occur on P. notatum roots grown in a soil of pH 4.9 (Dgbereiner, 1966). Root exudates from Paspalurn notatum have been shown to stimulate the growth of A . paspali (Machado and Dobereiner, 1969). A number of strains of this organism were unable to reduce nitrate (Pena and Dobereiner, 1974). Nitrogenase activity in these organisms was not inhibited by concentrations of NO3- up t o 2 0 mM. The derepressin of nitrogenase in NH4’-grown cells was the same in the presence of 10 mM nitrate and without added nitrate (Dobereiner and Day, 1975). Nitrite at a concentration of 0.1 mM inhibited growth of Azotobacter and at a concentration of 1.0 mM inhibited nitrogenase activity of Nz-grown cells (Dobereiner and Day, 1975).

C. Spirillum lipoferum

Spirillum lipoferum will be discussed in more detail because we consider it to be the most important bacterium in relation to N2 fixation in tropical grass-bacterial associations. 1. Taxonomy Beijerinck described this organism in 1923 as Azotobacter spirillum but later renamed it as Spirillum lipoferum (Beijerinck, 1925). Bergey ’s Manual (1957) contains a summarized description of the organism within Spirillaceae with the statement, “fixes atmospheric N2 in partially pure cultures, i.e., free from Azotobacter and Clostridium.” Schroder (1932) failed to find N2 fixation with one pure culture derived from a single cell isolate. Becking (1963) was able to

TABLE 1V Characterization of Spirillurn lipoferurn by Various Authors

D'dbereiner and Day (1976); Okon et al. (1976a); Sampaio ef al. (1976) Beijerinck (1925)

N, fixation in impure culture (mineral medium) N, fixation in pure culture (mineral medium) N, fixation in pure culture (mineral malate medium with yeast extract) N, fixation in pure culture (mineral medium with biotin) Growth on glucose Catalase Flagellation Pigment on nutrient agar Cell form in N free culture Cell form in nutrient broth NO,- to NO, NO, to gas

+

Schroder (1932)

+

Becking (1963)

N.S.~

I i

I1

IIP

+

+

+

+ +

+

N.s.

N.s.

i

N.S.

N.s

+

N.s

+

+

N.s. Bipolar Yellow Curved rods, polymorph Spirillum

N.s.

+

-

+

Polar N.s. Curved rods, polvmorph Spirillum N.s. Ns.

Polar Pink Curved rods

N.s. Pink Curved rods, polymorph Spirillum

N.s. Pink Curved rods

N.S.

Polar (tuff) N.s. Curved rods Spirillum

+

+

N.s.

N.s.

aGroups I, 11, or 111 as characterized in the text. bNot stated.

Spirillum

+ ++

+ +

+ +

-

Spirillum

+

NITROGEN FIXATION IN GRASSES

17

show lsN2 incorporation with a “Spirillum or Vibrio,” which he found to be probably identical to Beijerinck’s Spirillum lipofemm. In the 1975 edition of Bergey’s Manual, S. lipofemm was not included because no culture was available at the time (Krieg, personal communication). However, in a recent review on Aquaspirillum taxonomy (Krieg and Hylemon, 1976) a detailed description of S. lipoferum has been included. The N2 -fixing bacterium isolated from Digitaria roots (Dobereiner and Day, 1976) was identified as S. lipofemm following the description in Bergey s’ Manual (1957) and the original description by Beijerinck (1925). Recent studies revealed, however, at least three groups among the many strains which are now available (Okon et al., 1976a; Sampaio et al., 1976). The main differences between groups I and I1 are use of glucose, vitamin requirements, weak or no catalase activity, and larger more numerous polymorph cells in the latter. Group I11 strains are distinguishable from those of group I by the lack of the capacity to reduce NOz- to gaseous nitrogen forms (see further discussion on denitrification in Section III,C,2,c). In Table IV the main characteristics given by the various authors are summarized. From there it seems that the organisms described by Beijerinck (1925) and Becking (1 963) were group I1 organisms while those used by Schroder (1932) might be still another form because yellow pigment has never been observed in our laboratory.

2. Physiologv

a. Oxygen Effects. One of the key aspects of N2 fixation by Spirillum lipoferum is the oxygen concentration. The nitrogenase from S. lipoferum is extremely sensitive to oxygen and yet oxygen is necessary for the generation of ATP. This is a common problem for all aerobic N2 -fixing bacteria, but most of them have developed efficient oxygen protection mechanisms to exclude oxygen from the site of nitrogen furation (Postgate, 1974). Spirillum Iipofemm has a very poor O2 protection mechanism and cannot grow in air using N2 as the nitrogen source. However, vigorous growth is obtained in air when NH4+is used as the N source (Okon et al., 1976b). In an oxygen-limited chemostat, steady state could be established under air as long as the oxygen concentration in the medium was near zero (Dobereiner, 1976b). A partial pressure of oxygen above 0.005 atm inhibited nitrogenase activity. There does not appear to be a conformational protection mechanism for S. lipofemm, because in the presence of chloramphenicol there is no recovery of nitrogenase activity after exposure to oxygen (Dobereiner, 1976b). On the basis of similar observations, Okon et al. (1977a) devised an Oxystat to maintain a constant pOz for the growth of large batches of S. lipofemm. A pOz of 0.005 to 0.007 atm was found t o be optimal in these experiments which yielded the shortest generation time (5.5 hours). Semisolid medium is the simplest method of ensuring proper oxygen concentrations for optimal S. lipofemm N2 fixation. The organism is very motile and

18

CARLOS A. NEYRA AND J . DOBEREINER

therefore able to migrate toward the region of optimal p 0 2 . At this point cells concentrate very densely forming a characteristic pellicle. Active movement of S. lipoferum in the direction of the optimal p 0 2 and the agglomeration of groups can be easily observed in wet mounts under the microscope. The tendency t o agglomerate in groups possibly represents some kind of oxygen protection mechanism because O2 is consumed faster. Once bacterial density and oxygen demand increase, the bacteria move closer to the surface. When such cultures are disturbed by shaking or simply breaking the pellicle, nitrogenase activity ceases and is not restored under chloramphenicol (Okon et al., 1976a). The oxygen sensitivity of other Spirillum species which do not fix N2 has been attributed t o problems in Fe assimilation (Bowdre et al.. 1976). Increased concentration of ferrous sulfate in the medium or chelated Fe increased N2-dependent growth in S. lipoferum (Dobereiner, unpublished data). b. Temperature and pH. Temperature requirements for growth of Spirillum lipoferum are generally considered high but no strains isolated from temperate regions have yet been compared with the tropical strains (Day and Dobereiner, 1976; Neves et al., 1975). The fastest growth of seven strains was observed between 32" and 36°C and nitrogenase activity was maximal between 33' and 40°C with a pronounced drop below 33°C. Almost no activity was observed at 10°C but at 17°C about half maximal activity was maintained during a period of 8 hours (Day and Dobereiner, 1976). Exposure to 42°C initially increased the activity but after 5 hours nitrogenase activity was strongly inhibited. Temperatures of 45°C completely inhibited nitrogenase activity in all strains within half an hour (Neves et al., 1976). However, two strains recovered most of the original nitrogenase activity after 19 hours at 28OC. Exposure for 4 hours to 45°C was lethal to all strains. The requirements of pH for nitrogenase in pure cultures (Day and Dzbereiner, 1976) and in cell-free extracts (Okon et al., 1977b) are very narrow with maximal activity occurring between 6.8 and 7.8. The occurrence of S. lipoferum in soil is also highly pH dependent (Dobereiner et al., 1976). c. Nitrogen Metabolism. Spirillum lipoferum appears to have the ability to participate in various processes of nitrogen transformation in nature (Neyra et al., 1977). Nitrate can be assimilated by all strains, and cultures grown in I0 mM NO3- do not show any nitrogenase activity until all NO3- is exhausted. Under oxygen-limited conditions NO3- can be dissimilated to NO2- and rapidly reduced t o gas in groups I and 11. Nitrite continues to accumulate for more than 40 hours in group I11 (Fig. 1). Under anaerobic conditions there is no growth (Neyra and van Berkum, 1977) but NO3- is reduced and may be used instead of O2 t o support nitrogenase activity. Spirillum lipoferum is the first microorganism known t o bring about both N2 fixation and dissimilation of NO3- to gas. Neyra et al. (1977) confirmed denitrification by the accumulation of N 2 0 under CzHz as observed for other denitrifying bacteria by Yoshinari and Knowles (1976) and Balderston et al.

19

NITROGEN FIXATION IN GRASSES GROUP I

5

'N

4

0 2

b

3

'.!

2

'.

m

0

z

I E

I

20

40

$0

HOURS INCUBATION

FIG. 1. Time course of nitrate utilization and nitrite accumulation in the three groups of SpiriZlum lipoferum strains. Cultures were grown in semisolid malate medium with 5 mM KNO, .Values are means of four strains, three replicates of each.

(1976). Two representative strains of S. lipoferum group I , grown in semisolid malate cultures under air and exposed to CzHz with 1.4 mM N03-, simultaneously produced CzH4 and N 2 0 (Neyra et al., 1977). Cultures exposed to CzH2 under He were still more active in N 2 0 production but nitrogenase activity was low. In liquid cultures at oxygen-limiting conditions nitrogenase activity was increased within 1 hour by the addition of 10 mM NO3-, and the time course of NO3- reduction was coincident with the time course of the stimulated nitrogenase activity (Neyra and van Berkum, 1977). These findings suggest that S. lipoferum, where it occurs in high numbers may have an important role in all but one (nitrification) process of nitrogen transformation in the soil plant system. Okon et al. (1977a) showed that in maize roots from inoculated field plots in Wisconsin, 60 to 90% of the bacteria present in roots were S. lipoferum. No such comparative counts were made under tropical conditions but numbers of S. lipoferum in maize and sorghum roots in the field were at least in the order of lo4 to lo6 cells per gram of roots without any inoculation (DGbereiner et al., 1976; van Berkum, personal communication). The proportion of denitrifying S. lipoferurn strains is about 50%, and both forms were isolated from roots and soils (Sampaio et al., 1976). Their role in the root where carbon substrates are available can be anything from fixation of N2, reduction of NO3- to NOz- which could be assimilated by the plant, to denitrification of soil or fertilizer NO3-. Furthermore, NO3- may be able to support N2 fixation under oxygen-limiting conditions as occurs after heavy rainfalls.

20

CARLOS A. NEYRA AND J. DOBEREINER

Addition of 0.25% NH4C1 to a liquid or semisolid medium enhanced bacterial growth but completely inhibited C2H2 reduction (Okon et al., 1976a). When the cells were supplied with NH4' aerobically, the doubling time of S. lipoferum was as short as 1 hour and this was five times faster than growth on N2 (Okon et al., 1977b). d. Carbon Metabolism. Unlike most other N2 -fixing bacteria, sugars are poor carbon substrates for S.lipoferum. Optimal growth and nitrogenase activities are obtained when S. lipoferum is grown on malate, succinate, lactate, or pyruvate. These organic acids enhance 0 2 uptake in both cell suspensions and cell-free extracts (Okon et al., 1976a,b). The respiratory quotients Qo, (N) for S. lipoferum are about 10 to 20% of those of Azotobacter but similar t o those of Rhizobium (Okon et al., 1976b). This confirms a lack of oxygen protection mechanism for nitrogenase as has been found for Rhizobium when grown in culture medium (Bergersen, 1976). Enhanced 0 2 uptake in cell-free extracts of S. lipoferum on the addition of tricarboxylic acid cycle intermediates, lactate or pyruvate, indicates that the TCA cycle is operative in this organism (Okon et al., 1976b). Sugars and sugar phosphates, e.g., glucose, fructose, galactose, glucose6-phosphate, and fructose-6-phosphate failed to enhance O2 uptake in cell-free extracts from 5'. lipoferum. Glucose supports growth and nitrogenase activity only in strains of group I1 (Okon et al., 1976a; Sampaio et al., 1976). Galactose supports slow growth of intact cells (Okon et al., 1976a; Day and Dobereiner, 1976). These results suggest that the glycolytic and pentose phosphate pathways of metabolism in groups I and I1 are of minor significance in S. lipoferum. No significant differences in O2 uptake were found in cell suspensions or cell-free extracts for N2- and NH.++-growncells (Okon et al., 1976b). e. Nitrogenase. Nitrogenase activity of cell-free extracts of S. lipoferum has now been extensively studied by the Wisconsin group. An ATP-generating system, dithionite, Mg2+,and anaerobic conditions are required for nitrogenase activity (Okon et al., 1977b). An additional requirement is manganese, which is necessary for an activation factor. This activation factor has been found to be necessaly also for the activation of Rhodospirillum rubrum Fe protein of nitrogenase (Ludden and Burris, 1977). Purified activation factor from R. rubrum enhanced S. lipoferum nitrogenase and a crude extract from S. lipoferum was capable of activating purified inactive Fe protein from R. rubrum. This indicates a close relationship between these nitrogenases (Okon et al., 1977b). Addition of Fe protein from Azotobacter uinelandii to inactivated (-18OC storage) S. lipoferum extracts restored the specific activity which was even five times higher than that of active crude extract indicating that a major part of the activity had been lost during preparation of the S. lipoferum crude extracts (Okon et aL, 1977b).

21

NITROGEN FIXATION IN GRASSES

Spirillum lipoferum extracts lost their nitrogenase activity rapidly upon storage at -18°C but storage of liquid nitrogen had n o effect. Addition of purified Fe protein from Azotobacter vinelandii, R. rubrum containing the activating factor, or Bacillus polimyxa all restored nitrogenase activity but Fe protein from Clostridium pasteurianum did not. Highest specific activities were 10 nmol C2H4 produced per minute per milligram of protein under optimal conditions (Okon et al., 1977b). Nitrogenase in cell-free extracts was saturated at pCzH2 0.04 atm and the apparent KM was 0.0036 atm (Okon et al., 1977b). In intact cells nitrogenase was saturated only at pC2Hz 0.1 5 atm with an apparent K M of 0.022 atm (Day and Dobereiner, 1976). This difference may be due to a diffusion barrier in intact cells. Incorporation of NH4' and regulation of nitrogenase synthesis in 5'. lipoferum follow a pattern similar to that described by Tubb (1974), Streicher et aZ. (1974), and Brill (1975) for several other Nz-fixing bacteria. Active glutamine synthetase is necessary for the derepression of nitrogenase. Excess NH4+ represses glutamine synthetase and nitrogenase synthesis of S. lipoferum (Okon et al., 1976b). Activities of glutamate synthetase (GOGAT) were higher in N2-fixing S. lipoferum than in NH4'-grown cells, and the opposite was true with glutamate dehydrogenase (GDH). f: Efficiency in N2 Fixation. Nitrogen fixation in pure cultures of Spirillum lipofemm has been confirmed by the incorporation of lsNz (Becking, 1963; Okon et al., 1976a). It can of course most easily be demonstrated by Kjeldahl analysis. An example of such an experiment is given in Table V. Acetylene reduction assays are useful for comparisons between treatments and qualitative tests. The C2H2:Nz conversion factor was close to the theoretical value of 3 in three experiments 4.0 k 0.37 (10 strains in malate medium), 3.3 k 0.38 (10 strains in lactate medium), and 2.7 k 0.13 (one strain at three malate concentrations) (Dobereiner et al., 1976). TABLE V Nitrogen Fixation by Spirillurn lipofelurn Estimated by Kjeldahl Analysis

Strains used" Six strains of group I Six strains of p o u p 11 Five strains of group 111

Total N, fixed/flask ( 4 545 413 584

2 2

2

30 41 31

mg N, fixed/g carbon substrate 2 1 f 1.5 24 2 2.4

29 f 1.6 ______

'Triplicate flasks for each strain with N-free semisolid medium (20 ml) containing malate (10 mg) and succinate (10 mg) were incubated for 10 days at 33°C. The nitrogen contents in blanks without malate or succinate incubated with the cultures were subtracted.

22

CARLOS A. NEYRA AND J. DOBEREINER

In an earlier paper (Dobereiner and Day, 1974), efficiencies in terms of NZ furedlg substrate oxidized were assumed to be extremely high (100 mg N/g substrate) but this has not been confirmed. Okon et al. (1977b) in their oxystat obtained maximal efficiencies of 12 mg N/g malate consumed. These values correspond to those usually given in the literature for aerobic Nz -fixing bacteria during logarithmic growth (Mulder and Brotonegoro, 1974). Values obtained with semisolid stagnant cultures are 10 to 25 (Okon et al., 1976a) and 10 to 50 (Table V, Day and Dobereiner, 1976). Similar high values were obtained in oxygen-limited chemostat cultures with Azotobacter chroococcum (Postgate, 1971). Possible reasons for the much higher substrate conversion efficiency in the Rhizobium symbiosis have been discussed earlier (Dobereiner, 1974). From the data of Okon et al. (1977b) it seems that oxygen deficiency limits bacterial growth more than it inhibits nitrogenase activity, i.e., the generation time was increased by 52% while the nitrogenase activity was decreased by only 8%when the oxygen concentration in the culture was reduced to half optimal. It has been shown also that bacterial growth stops immediately when oxygen is removed from the cultures although nitrate reduction and nitrogen fixation continues (Neyra and van Berkum, 1977). It is therefore possible that the higher efficiencies observed in semisolid cultures are due to less growth but comparably higher nitrogenase activity per cell. Azotobacter consumes 80 to 90% of the carbon substrates for respiratory protection of nitrogenase (Mulder and Brotonegoro, 1974). Spirillum lipoferum has no apparent oxygen protection mechanism, so higher efficiencies should be expected.

3. Ecological Distn’bution Schriider (1932) reported occurrence of Spirillum lipoferum in 74 out of 76 soil samples collected all over Germany and Becking observed it “regularly” in samples collected for a Beijerinckia survey in Africa. It seems strange that an organism that attracts attention under the microscope by its active cycling movements should not have been noticed more frequently before. We have confirmed now that, at least in the tropics and subtropics, S. lipoferum is a very common soil and root inhabitant. About 60% of all tropical samples contained the organism while only 11% of the samples from temperate climate regions were positive (Table VI). The much wider distribution in Europe reported by Schrijder (1932) was not confirmed. Among 48 soil samples from Germany, Sweden, England, Isle of Man, and Spain only four were positive, while soils collected concomitantly in Rio de Janeiro State, also under various types of natural vegetation showed 85% positive samples (Silva and Dobereiner, unpublished data). Wheat and grass roots collected in the subtropical wheat region of KOGrande do Sul showed 20% positive samples (Table VI).

23

NITROGEN FIXATION IN GRASSES TABLE VI Geographic Distribution of SpiriIlurn lipofemrn in roots and soils collected in various countriesa Grass rootsb

Origin of samples

Latitude

Europe United States Africa Colombia Brazil, tropical Brazil, subtropical

48-58' N 43-47'" 6-15"N 3"N 0-23" S 30"S

No. of samples

% Positive

-

11 58 43 61 22

62

45 54 926 226

samples-

Soil No. of samples

% Positive samples

48 6

8 17 79 62

53 192 -

~

%Summarized from Dijbereiner er ol. (1976 and unpublished data). bWashed root pieces (0.5 cm) or one loop of soil were used to semisolid malate medium, C, H, reduction being measured after 40 33°C. Cultures which produced more than 10 nmol C,H,/hour, lipoferurn pellicle and contained thick rods filled with lipid bodies, ments were considered positive.

inoculate 3 ml N-free hours of incubation at showed the typical S. and spirilliform move-

Important limitations to Spirillum lipofemm occurrence in soil are vegetation and pH. Soils from tropical forest and savanna in equilibrium (cerrado) only sporadically show the bacteria. When the land is cultivated, the incidence of S. lipofemm increases considerably under grasses (Dobereiner et al., 1976). This would be expected because equilibrium systems are seldom nitrogen limited and even legumes bear few nodules (Bonnier and Brakel, 1969). Once harvests and lixiviation remove nitrogen, conditions are created which are more favorable for biological nitrogen fsation (Dobereiner el al., 1976). Under these conditions soil pH becomes the major limiting factor and highly significant correlations with S. lipofemm incidence were obtained. Samples with pH as low as 4.8, however, could be positive when the A13+ concentration is low (Dobereiner, 1976~).When the effect of soil pH on S. lipofemm incidence in soil and on Panicum maximum roots from the same sites was compared it became apparent that occurrence of the organism in soil is much more dependent on soil pH than is its occurrence in roots (Dobereiner et al., 1976). Specific plant effects of the major grasses have been discussed in Section I1,A. Preliminary evidence suggests that among a number of tropical plants other than grasses only sweet potatoes, cassava, and bracken fern roots contained S. Eipofemm even if it was present in soil (Dobereiner, 1976~).Under temperate climate conditions S. lipofemm was found only sporadically in maize and wheat roots and some other grasses (Dobereiner et al., 1976; Okon et al., 1977a; Barber

24

CARLOS A. NEYRA AND I. DOBEREINER

et al., 1976). Spirillum lipoferum was isolated by Sloger and Owens (1976) from maize roots in Beltsville, Maryland, and by Smith et al. (1976b) from forage grasses in Florida. Not much progress has been made in specifying the localization of Spirillum lipoferum in the various roots. As discussed in Section 11, in Digitaria and maize the roots are infected and the bacteria can be found inside the roots. Counts of S. lipoferum roots obtained from inoculated field plants in Wisconsin showed 9.0 X lo6 organisms on unsterilized roots and 1.6 X lo6 in surface-sterilized, crushed roots (Okon et al., 1977a). High numbers of S. lipoferum on the root surface compare with the legume symbiosis where lo6 Rhizobium cells per millimeter of roots can be found on the root surface without contributing to NZ fvration (Franco and Vincent, 1976). Whether S. lipoferum restricted to the root surface or only those inside the roots contribute predominantly to the nitrogenase activity is not known. Additional evidence for an inter- or intracellular localization in Panicum maximum are the observations of infection of seedlings (Garcia et al., 1976) and the abundant occurrence of S. lipoferum in surface-sterilized roots in soils with low pH where S. lipoferum was not abundant in the soil (Db'bereiner et al., 1976). It seems that the roots maintain a favorable pH for the bacteria. 4. Inoculation of Grasses with Spirillum lipoferum The possibilities of increasing crop yields by inoculation practices are often overestimated, even in legumes. The major problem is to establish the selected strain in the rhizosphere and t o have it infect the roots. Date (1971) concluded that not more than 5% of the nodules are formed by the inoculated strains when promiscuous legumes are sown in the field, and yield increases are seldom observed. Nothing is known yet about S. lipoferum specificity especially with respect to the three groups. Establishment on the roots does not seem to be plant specific because strains isolated from Digitarkz in Brazil established on maize roots in Wisconsin (Dobereiner et al., 1976). But establishment of Rhizobium on legume roots is also nonspecific (Franco and Vincent, 1976) even in the very specific species. The difficulty of identifying whether a certain strain has been isolated from an active site on roots instead of from the root surface further complicates these studies. In greenhouse experiments both sterile and unsterile pot cultures showed increases in nitrogenase activity (Burris, 1976; Barber et al., 1976; Mourarbt et d.,1975) but in none of these experiments have significant dry matter or total nitrogen increases been obtained. Most of these experiments, however, have been made with maize, and healthy normal maize plants are not easy to grow in pots. Inoculation of maize (Burris et al., 1976; Sloger and Owens, 1976) and wheat (von Biilow and Db'bereiner, unpublished data) grown in the field was unsuccess-

NITROGEN FIXATION IN GRASSES

25

ful. On the other hand, the Florida group reported significant forage yield increases with Pennisetum americanum and Panicum maximum inoculated with a Spirillum lipofemm strain isolated from Digitaria (group I) in Brazil (Smith et al., 1976a,b). In these experiments the maximal effect of inoculation was observed with an addition of 40 kg N/ha. 5. Culture Techniques and Identification

Culture techniques for Spirillum lipoferum are not generally known and therefore the most recent developments will be discussed here. More details can be found in the literature (Day and Dobereiner, 1976; Dabereiner et al., 1976; Okon et al., 1976a,b). The most characteristic growth of S. lipoferum is obtained in small vials w h c h are about half full of a semisolid N-free mineral medium containing potassium malate, biotin, and pyridoxal (Dobereiner et al., 1976). In this medium the organism starts growing from the inoculation point downward in a thin veil or round balloon-like form. On the second day oxygen demands are so high that the pellicle has become established 1 or 2 mm below the surface and becomes white and very dense, fine, and undulating. After some practice S. lipoferum can be recognized with certainty among other N2 -fixing bacteria which form thicker and less dense pellicles. To check nitrogenase activity the vials are closed with rubber bungs and exposed to IS% C2Hz for 1 hour. Care must be taken not to disturb the pellicle because this stops nitrogenase activity immediately. A few grains of soil, 0.5-cm-long root pieces, or appropriate dilutions (for counts) are used as inoculant. Hundreds of microscopic examinations, in cultures forming the characteristic pellicle, always showed S. lipofemm with typical fat droplets and active spiraloid movements. Isolation of Spirillum lipoferum from enrichment cultures is very easy. After one further enrichment in semisolid malate medium, cultures are streaked out on plates containing the same medium with 50 mg of yeast extract added. This is necessary because S. lipofemm grows in air only with combined nitrogen. After 5 days, small, dry, white or pinkish, raised, round, or irregular dense colonies are picked and transferred to N-free semisolid medium again. If they do grow and form the pellicle they are streaked out on potato agar to check for purity. On these plates after 1 week, colonies of S. lipoferum are very characteristically more or less pink, irregular, or round, but always dense and with umbonate elevations. Counts can be made by the serial dilution technique with liquid mineral medium for dilutions and small vials with semisolid malate medium for growth (van Berkum and Dobereiner, unpublished; Okon et al., 1977a). Small amounts of NO3- (0.1 to 1 mM) are useful to ensure growth of single cells in the highest dilutions. Positive dilutions are considered to be those with a Spinllum pellicle and nitrogenase activity.

26

CARLOS A. NEYRA AND J. DOBEREINER

To obtain large amounts of cells grown in Nz,Okon et al. (1 977b) recommend oxystat cultures. These are large fermenter cultures sparged with air where 0 2 concentration and pH are automatically adjusted to be maintained at pOz 0.005 atm and pH 6.8. Large batches of S. lipoferum can also be grown on NH4' and used for inoculants, etc. Such cultures grow fastest with heavy air sparging and there is no need for Oz control. IV. Factors Affecting Nitrogen Fixation in Grasses

The identification of the factors which limit nitrogenase activity under field conditions and in vitro is essential for any attempt to find agriculturally viable practice which may increase biological Nz fixation in grasses. The best general approach has been presented by Balandreau (1975) who monitored weekly during one growing season, with in situ soil plant cores of maize and forage grasses, soil and air temperature, light intensity, photosynthesis, air humidity, and soil nitrogen content. From these measurements a model was proposed to improve interpretation of in situ nitrogenase activity assays. Weekly variations were mostly dependent on soil N (mineral) and humidity while oscillations during the day could be attributed to soil temperature and light energy variations. Computer analysis of the interference of minimal and maximal soil and air temperatures, nitrogen content, and soil humidity in Digitaria nitrogenase activity confirmed minimum soil temperatures and NH4+content in soil as the major limiting factors (Abrantes et aZ., 1975). A large amount of information is further available on the effects of individual factors.

A. SEASONAL AND DIURNAL FLUCTUATIONS

Nitrogenase activity has been found to fluctuate throughout the growth cycle of the plants, particularly in grain crops. In general, maximal activities are found during reproductive growth of the plant. In field-grown maize, two peaks of nitrogenase activity have been observed. The first peak is associated with silk emergence and a second peak appearing at the onset of grain filling (von Biilow and Dobereiner, 1975; Neyra et al., 1976). Conversely, very little nitrogenase activity is observed before tasseling of the plants and after mid-grain filling. In field-grown sorghum plants maximal activities were reported to occur at flowering (van Berkum and Neyra, 1976). In this case, the decline of nitrogenase activity was coincident with the beginning of grain filling. Maximum nitrogenase activity in rice was observed at the heading stage and then declined rapidly (Watanabe and Kuk-Ki-Lee, 1975). Because mature, medium thick roots are the most active, as demonstrated for Digitaria decumbens (Day and Dobereiner,

NITROGEN FIXATION IN GRASSES

27

1976), maize (von Bulow and Db'bereiner, 1975), and sorghum (van Berkum and Neyra, 1976), it does appear that a full establishment of the root system is necessary for maximal rates of N2 fixation to occur. On the other hand, it is very likely that competition for available photosynthate by the grain could be the cause for the observed decline of nitrogenase activity during the seed-filling stages. Similar patterns of N2 fixation with ontogeny of the plant have been reported for legumes (Harper and Hageman, 1972; Thibodeau and Jaworsky, 1975). Diurnal fluctuation patterns have also been observed for several grasses. A peak of nitrogenase activity has been observed around midday and in some species a second peak was observed during the night. This pattern was observed for Paspalum notatum and sorghum (Dobereiner and Day, 1975) and for Panicum maximum and maize (Balandreau, 1975). Rice plants did not show any peak at night (Balandreau et al., 1974). The night peak was attributed to hydrolysis of carbon storage products accumulated during the day and their subsequent translocation and exudation in the rhizosphere (Balandreau et al., 1974). In general, most of the nitrogenase activity computed over a 24-hour period occurs during the light period and it may reflect the dependence of nitrogenase activity in grasses upon available photosynthate, as is the case in symbiotic systems. Recent results indicate that malate metabolism may play a major role in the supply of energy for nitrogen futation (van Berkum and Neyra, 1976). Malate is a primary product of C 4 photosynthesis in some species, e.g., maize, sorghum, and sugar cane (Chollet and Ogren, 1975). Malate is also one of the best substrates for the growth of Spinllum lipofemm, whereas glucose is a poor substrate. Addition of malate or bicarbonate during preincubation doubled the nitrogenase activity of excised sorghum roots (van Berkum and Neyra, 1976). Addition of similar concentrations of glucose had no effect.

B. PLANT GENOTYPE

Use of the excised root assay has revealed a wide range of nitrogenase activities for maize genotypes. Mean nitrogenase activities of some S1 maize lines were 10 to 20 times higher than the original cultivar UR-1 (von Biilow and Dobereiner, 1975). Crosses between higher fixing versus less fixing maize cultivars showed significant heterosis effects (von Biilow et al., 1976). Genotypic differences have also been shown in Paspatum notatum, Pennisetum purpureum, and wheat (Dobereiner and Day, 1974, 1975; Day et al., 1975b; Dobereiner, 1976a). Larson and Neal (1976) presented evidence for a selective colonization of a genetically defined line of wheat by a facultative N2-fixing Bacillus sp. All these results reveal the importance of plant genotype for optimal associations and

28

CARLOS A. NEYRA AND J . DOBEREINER

suggest the possibility of improvement of Nz -fixing associations by plant breeding.

C. TEMPERATURE

Because of the relatively high temperature requirements for Spirillum lipoferum development, tropical areas appear more favorable for a higher incidence of this bacterium (Dobereiner et at., 1976). However, differences among plant species are expected to occur in relation to tolerance to relatively low temperatures. Optimal temperature (31°C) for nitrogenase activity of pure cultures of S. Zipofemm and of isolated maize roots were coincident (Dobereiner et al., 1975). Soil temperatures below 25°C were found to be a major limiting factor to nitrogen futation in roots of Digitaria decumbens cv. transvala (Abrantes et al., 1975). Nitrogenase activity of Spirillum lipoferum isolated from Digitaria roots was also inhibited by temperatures below 25°C. Although Spirillum strains isolated from maize and Digitaria did not show differences in nitrogenase activities at optimal temperatures, they seemed to behave quite differently at lower temperatures. At 22°C the maize strains were five times more active than the strains isolated from Digitaria (Neves et a l , 1975). Nitrogenase activity on forage grass roots is also very low once night temperatures fall below 18°C. This has been attributed to effects upon plant growth (DSbereiner and Day, 1974).

D. OXYGEN Because of the oxygen sensitivity of the nitrogenase enzyme system (Ljones, 1974; Postgate, 1974) and the known effects of oxygen on nitrogen fixation by bacterial cultures (Day and Dobereiner, 1976), it is not surprising that optimal nitrogenase activities on the roots of forage grasses are found at pOz far below that of air. Studies on the Paspalum notatum-Azotobacter paspali association revealed that nitrogenase activity on the roots was extremely sensitive to changes in pOz. Nitrogenase activity was almost completely inhibited in air or in the absence of Oz and was greatest at p02 0.04 atm (Dobereiner et al., 1972a). In contrast, the activity of soil cores containing P. notatum plants was little affected by changes in pOz above the soil (Dobereiner et al., 1972a). Very little is known about the oxygen protection mechanism in the intact soil grass system. It is known, however, that Spirillum lipofemm has a very poor oxygen protection mechanism for its nitrogenase (Day and Dobereiner, 1976; Okon et aZ., 1976a). This would explain the oxygen sensitivity of the nitrogenase activity on the roots of tropical grasses in association with this bacterium. In Digitaria, maize, and sorghum roots maximal activities are found at p 0 2 0.01 to 0.02 atm

NITROGEN FIXATION IN GRASSES

29

and pure cultures of Spirillum lipoferum show the same pOz requirements as the isolated roots (Day and Dobereiner, 1976; Abrantes et al., 1975). Roots preincubated under a p 0 2 of 0.02 atm gave maximal activities without additional supply of O2 at the time acetylene was injected into the vials.

E. COMBINED NITROGEN

High levels of combined nitrogen in the soil or the application of heavy nitrogen fertilization reduce the potential for nitrogen fixation in grasses. Exposure of excised roots to NO3-, NOz-, or NH4' during the preincubation period inhibited nitrogenase activity in sorghum and maize roots (van Berkum and Neyra, 1976; Neyra et al., 1976). In sorghum roots a 50% inhibition was observed at 1 0 mM NO3- while a similar effect was obtained by an exposure to 0.1 mM NH4+ or NOz-(van Berkum and Neyra, 1976). These results indicate that the level of NH4+ in the soil solution may be a limiting factor for the development of nitrogenase activity in grasses. T h s has been confirmed in experiments conducted with Digitaria decumbens cv. transvala. Ammonium concentrations in the soil solution above 200 ppm severely inhibited nitrogenase activities (Abrantes et al., 1975). From the multiple regression equation for NH; and soil temperature (r = 0.75) it was calculated that at 27°C (soil temperature) in the absence of NH4' or at 32.5"C in the presence of 50 ppm NH4' (soil water basis) a nitrogenase activity of 50 nmoles CzH4 per gram of dry root per hour, could be obtained. Nitrogenase activities of Pennisetum purpureum and Digitaria decumbens roots, determined throughout the season, were not reduced even after eight applications of 20 kg N per ha as NH4N03 (Dobereiner and Day, 1975). Balandreau et d. (1975) reported that upon the application of different amounts of (NH4)2S04 at sowing there was a marked effect on the nitrogen fixation in rice seedlings. For additions of up t o 40 ppm, Nz fixation was stimulated, but higher applications resulted in a marked decrease. It was assumed that the slight increase of nitrogenase activity at low nitrogen levels could be attributed to an increase of root exudate (Balandreau et al., 1975). Watanabe and Kuk-Ki-Lee (1975) reported that NPK fertilizer appears to promote heterotrophic Nz fixation in paddy rice, while phototrophic N2 fixation appears to be predominant in nonfertilized soils. These observations are of great importance because at low levels of combined nitrogen in the soil the simultaneous utilization of biological Nz fixation and mineral N fertilizer may be possible. This possibility is further illustrated in Fig. 2. The addition of 40 kg N/ha at planting allowed the development of nitrogenase activity in maize roots while reasonable nitrate reductase activities (in vivo assay) were determined in the leaves of the same plants (Neyra et al., 1976).

30

CARLOS A. NEYRA AND J. DOBEREINER 1

Days after Germination FIG. 2. Seasonal variations of nitrogenase and nitrate reductase activities in field-grown maize plants. Nitrogenase activity was determined in excised preincubated roots and nitrate reductase activity (in vivo assay) in leaf blades. Nitrogenase activity under low N (40 kg/ha) (0-0) and high N (200 kg/ha) (0-0) fertilizer. Nitrate reductase activity of low N (40 kg/ha) treatment (n---o). Silk emergence (1) and mid-grain filling (2) stages are indicated by arrows.

Similar results were obtained with field-grown sorghum (van Berkum et al., 1976). However, heavy doses of fertilizer (200 kg N/ha) severely inhibited nitrogenase activities in both maize and sorghum plants which illustrates the fact that, in areas receiving continuously high doses of nitrogen fertilizer, the potential for N2 fixation may not be realized. V. General Discussion

Several lines of evidence presented throughout this review have shown the existence and operation in nature of grass-bacteria associations able to bring about N2 fixation. Several observations also indicate that these associations are contributing significantly to the N economy of the plants. However, the actual contribution of Nz furation to plant N is not known, as "N studies of the amount and rate of transfer of the fixed nitrogen to the host plant are lacking (Day et aL, 1975a). Nevertheless, nitrogen fixation in Digitaria decumbens and Paspalum notatum cores has been confirmed by the incorporation of "Nz (De-Polli et al., 1976).

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Acetylene reduction assays over a 24-hour period in cores indicating fixation of more than 100 g N/ha per day have been obtained with several tropical forage grasses, rice (algal fixation substracted), and occasionally with wheat and rye grown under tropical conditions (Dobereiner, 1976a,b; Watanabe, 1976). In most instances, a significant correlation of core assays with preincubated excised root assays has been obtained, with the latter usually underestimating (Dobereiner, 1976b). On the other hand, N2 furation estimated by the excised root method in maize and sorghum has been shown to be several-fold higher than the amount of N2 fixation obtained from core assays (Barber et al., 1976; Tjepkema and van Berkum, personal communication). These results suggest that the excised root assay does not provide a reliable measure of N2 fixation in maize and sorghum (Barber et al., 1976). However, the possibility of underestimation by the core assays has not been ruled out as yet. The major argument against the excised root assay is that all grasses assayed by this method show a lag period (8 to 18 hours) before acetylene reduction starts (Dobereiner et al., 1972a; Abrantes et aZ., 1975; Dobereiner and Day, 1975). All attempts to eliminate this lag have been unsuccessful (Dobereiner and Day, 1975). Thus, in order t o obtain linear rates of acetylene reduction, a preincubation overnight at low O2 tensions (2% 0,) appears necessary. Nevertheless, recent reports (Sloger, 1976; Sloger and Owens, 1976) indicate that high rates can be obtained in freshly harvested surface-sterilized maize roots (up to 1000 nmolds C2 H4/hour per gram of dry root). Still in other assays a 20-fold increase in activity was observed after an overnight preincubation at 2% 0 2 .Although the lag has been observed by several workers, no satisfactory explanation has been forthcoming. Barber et al. (1976), Okon et al. (1977a), and van Berkum (unpublished data) working with excised roots of maize and sorghum, have. observed a substantial increase, during the preincubation period, in numbers of Nz -fixing bacteria capable of growing in malate, which could explain the higher nitrogenase activities observed after preincubation of these plants. On the other hand very high numbers of S. lipoferum have been found on young maize roots and on roots from nitrogen-fertilized plots which exhibited no nitrogenase activity (Podesta et aZ., 1976). Therefore, higher numbers of Nz-furing bacteria do not necessarily mean higher fixation. There might also be differences according to the localization of the bacteria outside or inside the roots. A great deal of information and insight into the field of Nz fixation in grasses has been obtained using the excised root assays. Because this method allows the handling of large numbers of samples from the field and the good reproducibility of the results, it has proven to be a valuable tool for investigating many aspects of N, fixation in grass-bacterial associations, such as the distribution of Nz-fixing associations among different plant species, as well as plant genotype and physiological studies. The seasonal pattern of nitrogenase activity associated with plant ontogeny and the genotypic differences observed with several plants show that the physiology of the host can control the level of nitrogenase activity

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of the bacteria associated with their roots. Furthermore, the high degree of specificity of certain associations as in Paspalum notarum-Azotobacter paspali (Dobereiner and Campelo, 1971) or the highly specific association of an Nz-fixing Bacillus sp. with a genetically defined line of wheat (Larson and Neal, 1976) clearly indicate the potential of plant breeding as a promissory tool for achieving better plant-bacteria associations with a greater Nz-fming ability. The participation of Spirillum lipofemm in all but one (nitrification) of the steps in the nitrogen cycle and its widespread distribution in soils and roots makes this organism very useful for studying the various aspects of nitrogen transformation in nature. While some strains of S. Zipofemm fur Nz and denitrify (groups I and II), others do not denitrify (group 111). Studies of methods to optimize N2 fixation and minimize denitrification are of obvious importance. In view of the existence of three different groups of S. Zipofemm, the exact role of this organism in the overall nitrogen metabolism in the soil-plant system needs to be studied in more detail. While biological Nz fixation could be sufficient for the maintenance of forage grasses growing in their natural habitat, it is unlikely that biological N2 fixation alone could satisfy all the nitrogen requirements of high yielding agricultural crops and, therefore, studies on the interaction between combined N and biological N2 assimilation should be ranked as a high research priority. We know that Spirillum lipofemm grows well in the presence of NO; or NH4+ (Okon et aZ., 1976a; Neyra and van Berkum, 1977) but nitrogenase is inhibited. On the other hand, NO3- is usually the main form of nitrogen in well-aerated soils, where conditions are favorable for nitrification. Therefore, it is likely that under field conditions NO3- will limit Nz fixation. The search for strains which fix Nz in the presence of NO3- has good possibility of success in the near future. Development of nitrate-reductase deficient mutants of S. Zipofemm as it has been shown for Rhizohium (Sik et QZ., 1976; Gibson, personal communication) should be possible. Although good progress has been made in understanding the importance of environmental and plant factors, the exact nature of the grass-bacteria associations is stdl unclear. Recent observations on other N2-furing plant-bacteria associations might help to advance a working hypothesis for studies of this kind. Rhizobium can now be grown in culture media and produce active nitrogenase (Pagan et al., 1975; Kurz and La Rue, 1975) but combined nitrogen is still necessary for growth (Bergersen, 1976; Keister, 1976). The fixed nitrogen is excreted as NH4+into the medium and partially reassimilated (Bergersen, 1976). In the nodules, bacteroids excrete the fixed Nz as NH4' which is assimilated by plant enzymes (Scott et al., 1976). Ammonia does not repress nitrogenase synthesis in free-living rhizobia grown either on agar (Kurz and La Rue, 1975) or in shake cultures under microaerophilic conditions (Keister, 1976). More similar still to the grassSpiriZlum association are the symbioses of blue-green algae where the algae are able both to fur Nz in vitro and to associate

NITROGEN FIXATION IN GRASSES

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with plants. Examples are several lichens and the liverwort symbioses. Stewart (1976) reported profound changes in the physiology of these algae when they are living in symbiosis with the procaryotic macrophyte: The percentage of heterocysts increased, photosynthesis ceased, and NH4’-assimilating enzymes were inhibited and therefore the algae excreted the fured Nz as NH4+ and the efficiency (nmol C2H4/mg protein) increased by a factor of 3 to 7. Algae separated from the macrosymbiont needed more than 24 hours to reassume growth. On the basis of these observations Stewart (1976) suggested that the restriction of Nz-fixing symbioses to a few species is linked to the necessity of a plant factor which switches the bacterial NH4+ assimilation pathway off. How far these findings apply to the grass associations remains to be seen.

ACKNOWLEDGMENTS We acknowledge the support of the Program for International Cooperation in Training and Basic Research on Nitrogen Fixation in the Tropics, sponsored by the Brazilian National Research Council (CNPq), the Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA), and the Universidade Federal Rural d o Rio de Janeiro. We wish to thank Dr. D. B. Scott for helpful discussions and revision of this manuscript and Mrs. C. Scott for helpful assistance.

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van Berkum, P., Neyra, C. A., and von BUlow, J. F. W. 1976. Reuniao Bras. Milho Sorgo, I I th, Piracicaba, S2o Paulo. Vancura, V., Abd-El Malek, Y., and Zayed, M. N. 1965. Folia Microbiol. (Prague) 10, 224-229. Venkataraman, G. S. 1975. In “Nitrogen Fixation by Free-Living Micro-Organisms” (W. D. P. Stewart ed.), pp. 207-218. Cambridge Univ. Press, London and New York. Verdade, F. C. 1967. Proc. Biol. Ecol. Nitrogen (IBP Conf.), Davis, Calg Vlassak, K., and Jain, M. K. 1976. Environ. Role Nitrogen-Fixing Blue-greenAlgae Asymbiotic Bacteria, Int. Symp., Uppsala. von Bulow, J. F. W. and Dgbereiner, J. 1975. Proc. Natl. Acad. Sci. U.S.A. 72,2389-2393. von Biilow, J. F. W., DGbereiner, J., and Podestil, J. A. 1976. Reuniao Bras. Milho Sorgo, I 1 th, Piracicaba, Srlo Paulo. Watanabe, I. 1975. Res. Results Rep., Soil Microbiol. Int. Rice. Res. Inst. (IRRI), Los Banos, Laguna, Philippines. Watanabe, I. 1976. Environ. Role Nitrogen-Fixing Blue-green Algae Asymbiotic Bacteria, Int. Symp., Uppsala. Watanabe, I., and Kuk-Ki-Lee. 1975. Int. Symp. Biol. Nitrogen Fixation Forming Syst. Humid Trop., Int. Inst. Trop. Agric, (IITA),Ibadan, Nigeria. Yoshida, T. 1971a. Soil Sci. Soc. Am., Proc. 35, 150-157. Yoshida, T. 1971b. Res. Results Rep., Soil Microbiol. Int. Rice Res. Inst. (IRRI), Los Baiios, Laguna, Philippines. Yoshida, T., and Ancajas, R. R. 1973. Soil Sci. Soc, Am., Proc. 37,42-46. Yoshinari, T., and Knowles, R. 1976. Biochem. Biophys. Res. Commun. 69, 705-710.

NOTE ADDED IN PROOF While this paper was in press, Krieg (Krieg, N. R. 1977. Conf. Genet. Eng. forNirrogen Fixation, Brookhaven Nat. Lab., Brookhaven, New York), based on extensive DNA base c o m p e sition studies with 65 Spirillum lipoferum strains isolated from around the world, proposed reclassification of the organism as Azospirillum lipoferum (group I1 stated in Table IV) and Azospidum brasilense (groups I and I11 stated in Table IV).

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W R .Scowcroft Division of Plant Industry. CSIRO. Canberra. Australia

I. Introduction .................................................. I1. Plant Cell Tissue Culture ......................................... A. Methodology ............................................... B. Freeze-Preservation of Cell Cultures .............................. C. Plant Regeneration from Cell Cultures ............................ I11. Anther Culture and Haploids ...................................... A . Methodology ............................................... B. Theory and Application ....................................... IV. Mutant Isolation and Selection .................................... A . Amino Acid Analogue-Resistant Mutants .......................... B . Disease-Resistant Mutants ...................................... C. Stress-Resistant and Other Mutants .............................. V. Plant Cell Protoplasts ............................................ A . Methodology of Isolation ...................................... B . Protoplast Culture ........................................... C. Protoplast Fusion and Somatic Hybridization ...................... D Limitations to Somatic Hybridization ............................ VI . Genetic Transformation in Plants ................................... A . Modification by Homologous DNA .............................. B . Modification by Heterologous DNA .............................. C. Molecular Gene Manipulation ................................... D . Plant Improvement and Desirable Genes for Manipulation . . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I

39 40 40 42 42 44 44 46 48 50 52 53 55 55 56 58 60 61 62 62 65 70 73 74

Introduction

Plant improvement has its origins in man’s prehistory . The perception and intuition of our ancestral plant breeders gave us the majority of our modern day agricultural species and certainly those species of major agricultural importance . Within this history. the relatively recent knowledge of genetics has provided some understanding of what were purely empirical observations and. indeed. has improved the efficiency whereby a plant breeder reaches his objective . Plant breeding is a continuing enterprise . The unrelenting demand for increased food production. alteration in the spectrum of disease pathogens. and changes in economic and consumer demands ensure the continuance. and hopefully increased efficiency. of this enterprise . 39

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A plant improvement program has a set of defined objectives. The success of the plant breeder depends first on his ability to define and assemble the requisite genetic variability he considers necessary to meet this end. H i s second task is to recombine this genetic variability and extract from the gene pool those gene combinations which yield superior genotypes according to his objectives. Although a given plant improvement program may have specific limitations, there are two general constraints which limit the rate at which objectives are achieved. First, most plant breeding involves recurrent cycles of hybridization and selection, usually under field conditions. This is time- and resource-consuming despite the development of ways to lessen the interval between one cycle and the next. Second, the spectrum of genetic variability is usually restricted to the species with which the breeder is concerned. There are of course notable exceptions to this latter constraint as in breeding for rust resistance in wheat, disease resistance in tobacco, or the development of triticale. Developments in basic genetics have partially alleviated one or both of these constraints. The ability to manipulate chromosomes, the use of polyploidy, the induction of mutations, and the theory of quantitative inheritance have each contributed to the plant breeder’s armory. During the 1960s the immense knowledge gained from molecular biology and genetics was arguably presented as providing a panacea for the world’s biological ills. The then infant science of plant cell culture was seen as a vehicle for translating the conceptual, and in some cases experimental, methodology of molecular biology to plant biology, and ultimately to plant improvement. This review examines recent developments in plant cell culture and genetics, particularly in the context of their possible role in plant improvement. The potential value of these developments is founded on the basis that plant cells can be cultured under defined conditions, biochemical mutants can be isolated, somatic hybrids can be obtained by protoplast fusion, haploid cell lines and/or plants can be obtained, and cell cultures can be induced to regenerate fertile plants. The last feature allows genetic modifications at the cellular level to be evaluated in mature plants, and it is for this reason that plant cell culture may be of value to plant improvement. Most of this review will be concerned with aspects of cell culture and genetic manipulation. However, some aspects are so closely related to whole plant studies that if they were excluded this review would be incomplete. I I. Plant Cell Tissue Culture

A. METHODOLOGY

Plant cells have the enormous advantage of totipotency. Haberlandt predicted this as early as 1902 (cf. Vasil and Hildebrant, 1967) but almost 60 years

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elapsed before a mature plant was regenerated from a single cell (Braun, 1959). The ability t o culture plant cells under defined conditions stems from the efforts of Gautheret (1939) and White (1942). Since then many research workers have examined and refined the nutritional and environmental requirements of plant cells in culture. Particular mention must be made of the work of Skoog and co-workers (Murashige and Skoog, 1962; Linsmaier and Skoog, 1965) from which most currently used tissue culture media have been derived. Plant tissue culture media consist of inorganic salts, trace elements, vitamins, a carbon source for energy, and plant growth regulators. Although generally not essential, undefined organic supplements such as yeast extract, casein hydrolysate, plant extracts, and the liquid endosperm of coconuts are often used as supplements. The basic methodology of plant culture has been adequately covered (Kruse and Patterson, 1973; Street, 1973; Gamborg and Wetter, 1975) and will not be further elaborated here. A very large number of plant species representing many families of gymnosperms, dicotyledons, and monocotyledons have been successfully cultured, and the list continues t o expand. Virtually any part of a plant can be induced to form callus including embryos, root or stem sections, hypocotyl, cotyledons of immature seeds, and germinating seedlings and leaves. The use of young plants or rapidly growing plant tissue is preferable since nonpathogenic bacteria tend to invade older plants and quiescent tissue. Cell cultures of many species have been initiated in our laboratory, including dicotyledons, monocotyledons, and herbaceous perennials. Although seeds are initially slower to callus than, say, stem internode segments, most success has been achieved by initiating callus from seeds germinated directly on the culture medium. The rigorous surface sterilization that can be applied to seeds results in minimal contamination of the resultant callus cultures. Plant cells can be cultured on agar media in shaken liquid suspension cultures or in continuous culture fermentors. Cell doubling time varies from 15 hours to several days. Suspension cultures are particularly useful for genetic experiments because they generally grow more rapidly than agar cultures and provide large populations of physiologically homogeneous cells. Moreover, several methods are now available for adequate and repeatable measurement of growth rate. These include dry cell weight, packed cell volume, direct cell counting, and turbidity measurements. The use of this latter measure of cell growth of tobacco cultures has provided us with repeatable estimates of growth. Plant cell cultures tend to be asynchronous during growth. This tends not to be a problem in genetic studies but most certainly is in related studies on the cell cycle. Techniques to induce synchrony include the use of inhibitors of DNA synthesis or of mitosis, nutrient starvation, exclusion of kinetin (cf. Yeoman, 1974), and the partial replacement of the aerobic gas phase with nitrogen (Constabel et al., 1974).

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W.R. SCOWCROFT B. FREEZE-PRESERVATION OF CELL CULTURES

A recent development is the ability to store plant cell cultures by freezepreservation. This technique is of particular value to cell culture genetics, since any genetic program will sooner or later have a battery of cell culture mutants which need to be stocked. Indeed such a technique is essential for callus of those species which cannot be regenerated to produce mature plants and hence seed. Freeze-preservation of cell cultures has also been advocated as a means of preserving valuable genetic stocks of asexually reproducing species of agricultural importance (Henshaw, 1975). The most extensive studies on freeze-preservation have been done with carrot cells (Nag and Street, 1973, 1975a,b) but some studies have been extended to include Acer cells and shoot apices of carnation (Siebert, 1976). The procedure involves an initially controlled and slow rate of freezing prior to transfer to liquid nitrogen at -196°C. A cryoprotectant such as glycerol of dimethylsulfoxide is required during the freezing process. A high rate of recovery results if the thawing process is fairly rapid and the cryoprotectant is quickly removed by washing (Nag and Street, 1975b). Freeze-preservation is a routine aspect of animal cell culture research. Undoubtedly this will also apply in the plant cell tissue culture research.

C. PLANT REGENERATION FROM CELL CULTURES

From the genetic and plant improvement aspect, the ability to regenerate fertile plants from cell cultures means that genetic manipulations at the cellular level can be evaluated in mature plants and possibly utilized in conventional breeding programs. The totipotency of plant cells has been invaluable in research which seeks to understand the process of embryogenesis (Street and Withers, 1974). Eventually this might provide a set of principles so that plant regeneration from cell cultures can be achieved at will. Unfortunately, nature has so far failed to yield her secrets. Consequently, attempts to regenerate plants from cell cultures must be approached empirically. Regeneration has been attempted for a very diverse number of plant species and many notable successes have been achieved. The experimental inputs into these studies have been evaluated by Murashige (1974) in an attempt to decipher the important variables in the process of plant regeneration. While Murashige (1 974) was able to show that a number of nutritional and physical parameters can affect the process of plant regeneration, the major breakthrough of general applicability was that of Skoog and Miller (1957). They showed that the relative concentration of the growth regulators, auxin and cytokinin, determined the pattern of organogenesis. A relatively high ratio of cytokinin to auxin suppressed root formation and enhanced shoot initiation. The converse favored root formation over shoot development. This finding has more or less been systematized to give combina-

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tions of various concentrations of representatives of these two classes of growth regulators (e.g., de Fossard et al., 1974; Kartha, 1975; Kartha et al., 1976). An aspect of plant regeneration which has become apparent is that there is significant genetic variability affecting the ease of plant regeneration from callus. In a number of studies on regeneration of plants from callus of species such as tomato (Gresshoff and Doy, 1972), Brassica oleracea (Baroncelli e t al., 1973), maize (Green and Philipps, 1975), and alfalfa (Bingham et al., 1975) several varieties were used. In each case the capacity to regenerate plants differed between varieties. We have had a similar experience with barley, where only two of 30 varieties tested were successfully regenerated into plants. More recently, Bingham et al. (1975), by recurrent selection, have developed a line of alfalfa which regenerates at very high frequency. After only two cycles of recurrent selection the frequency of regenerating genotypes increased from 12 to 67%. This indicates that regenerative capacity is highly heritable. In attempts to develop tissue culture and regeneration for a particular species, most workers tend to utilize a limited number of varieties (or genotypes) and vary the culture media and growth regulator concentrations. The alternate option is to restrict the variability in the culture media and utilize diverse genotypes. For particularly important agricultural species, benefit in terms of ease of regeneration would most certainly be derived from a few cycles of recurrent selection for plant regeneration. The number of plant species in which the potential for plant regeneration from callus has been demonstrated is impressive. The recent compilation of species by Murashige (1974) even now can be considerably expanded. The majority of successes have been achieved with exotic and horticultural species but, among the agricultural gramineae, plants can be regenerated from callus of wheat (Shimada et al., 1969), rice (Nishi et al., 1968, 1973), sugar cane (Nickel1 and Heinz, 1974), maize (Green and Philipps, 1975), barley (Cheng and Smith, 1975), and oats (Lorz ef al., 1976). Success has also been achieved in regenerating plants from callus of coffee (Herman and Hass, 1975), cassava (Kartha and Gamborg, 1975), potato (Skirvin et al., 1975), and for some tree and shrub species (Sommer et al., 1975; Pierik, 1975). Until recently the legumes have proven recalcitrant to differentiation. Despite extensive research with soybean cell cultures, the differentiation of mature plants from callus has not been reported. The two legume species where plants can be regenerated from callus with ease are alfalfa (Bingham et al., 1975) and a tropical pasture legume, Stylosanthes hamata (Scowcroft and Adamson, 1976). It is noteworthy that S. hamata is a perennial legume, and that the more successfully regenerating varieties of alfalfa were creeping rooted types which develop adventitious shoots from root cells. Apart from the “mere” difficulties of translating the potentialities of plant regeneration into everyday reality there are other limitations. Among these the maintenance of long-term embryogenic potential is paramount (Murashige,

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1974; Thomas and Davey, 1975). Street (1975) and Sheridan (1975) have discussed the loss of regenerative capacity as a function of an increase in aneuploidy. Some cell lines of Haplopappus, Crepis, and Lilium do not tend toward aneuploidy and retain long-term embryogenic potential. The cellular cloning of plant species is used in the asexual propagation of many species (Murashige, 1974) and in the production of “disease-free’’ plants. In the ensuing discussion of the genetic modification of plants at the cellular level, the ability to regenerate plants from such cells is essential for such modification to be of value to plant improvement. First, the cellular modification must be evaluated in the mature plant so that the plant breeder may judge their potential contribution to the gene pool. Second, the plant breeder must be able to inject such novel genotypes into his breeding population by conventional sexual hybridization for which he needs mature fertile plants. Ill. Anther Culture and Haploids

The potential value of plants with the gametic number of chromosomes (haploids) in basic and applied genetic research, has been recognized ever since Blakeslee et al. (1922) first described a haploid mutant in Datura. The semantic arguments surrounding the use of the term “haploid” are discussed by de Fossard (1974). Because of the great potential to plant improvement, advances in haploid plant research have been closely monitored. Kimber and Riley (1963) reviewed the then known origins of haploidy. They listed 71 species in which haploids had occurred spontaneously, as a consequence of hybridization, from twin embryos or by experimental induction. More recent progress was considered in depth at a conference in Canada in 1974 (Kasha, 1974). The production of haploids through parthenogensis or by chromosome elimination in barley (Kasha and Kao, 1970) and recently in wheat (Barclay, 1975) have as much potential importance to basic and applied genetic research as those generated by anther culture. I will not discuss the former two methods further (cf. Kasha, 1974), but acknowledge that the practical and theoretical consequences of producing haploids by anther or pollen culture also apply to these other methods.

A. METHODOLOGY

The technique of producing haploid genotypes by anther culture was pioneered by Guha and Maheshwari (1964) in DQmra. This methodology was developed by many, but most notably by J. P. and C. Nitsch and co-workers (Nitsch and Nitsch, 1969; Nitsch, 1972). More recently Nitsch (1974, 1975) has

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demonstrated that isolated microspores of tobacco can be cultured to produce haploid plants. Extensive cytological analyses in Nicotiana and Datura have elucidated the events which lead to the production of haploids (see Sunderland and Dunwell, 1974; Sunderland, 1974). Under appropriate culture conditions the normal process of pollen development is arrested between the tetrad stage and the conclusion of the first pollen mitosis. Subsequently the generative cell degenerates while the normally quiescent vegetative cell divides to form an embryolike structure. Cytological events indicate that haploidy follows one of either of two developmental pathways, each of which leads to the ultimate degeneration of the generative partner (Sunderland, 1974). Following induction of cell division in the pollen, embryos develop which give rise to plantlets actually growing out of the anther, or callus may be formed which then has to be differentiated to regenerate a plant. The former sequence is characteristic primarily of tobacco and Datura, and the latter is the more general consequence in other species. In cases where callus, hopefully with a haploid complement, is formed, controlling the ploidy level in the callus may be a serious limitation to its practical use for generating haploids. On the basis of the use of p-fluorophenylalanine (PFP) to increase frequency of haploid segregation in fungi (Day and Jones, 1971), Gupta and Carlson (1972) claimed that PFP inhibited the growth of diploid, but not haploid, cells of tobacco. This claim has not been sustained (Zenk, 1974; Dix and Street, 1974) or at best is not very reproducible (Chaleff and Carlson, 1974). An additional problem, as pointed out earlier, is the difficulty of regenerating plants from callus. This varies from species to species, and indeed from genotype to genotype within a species. With haploid callus I would judge that the problem of regenerating a representative sample of haploid genotypes might be even more difficult. Notwithstanding, haploid plants have been successfully produced in species other than tobacco and Datura. Catalogs of species in which haploids have been produced are provided by Smith (1974), McComb (1974), and Sunderland (1974). Although specific reports cannot be cited, a haploid information exchange service, edited by the Haploid Project Group, Max Planck Institute fur Biologie, Rosenhof, Germany (see Kasha, 1974, p. 41 l), carries reports of both successful and unsuccessful attempts to induce haploids. Apart from tobacco, other important crop species in which haploids have been produced by anther culture include rice (Niizeki and Oono, 1968; Oono, 1975; Wang e t al., 1974; Laboratory of Genetics, 1975; Woo and See, 1975), wheat (Ouyang et al., 1973; C. Wang et aZ., 1973;Picard and de Buyser, 1973), barley (Clapham, 1973; Dale, 1975), triticale (Y. Wang et al., 1973; Sun et al., 1974), tuberous Solanum species (Irikura and Sakaguchi, 1972; Dunwell and Sunderland, 1973), and turnip rape (Brassica campestris) (Keller e t al., 1975). The use of haploids in plant improvement, or indeed any research where a gametophyte is required from the haploid sporophyte, requires that the chro-

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mosome complement be doubled. Fortunately, chromosome doubling techniques are already efficient. An excellent account of their status and methodology is given by Jensen (1974).

B. THEORY AND APPLICATION In a breeding program designed to produce pure line varieties in a self-fertilized species, or inbreds for hybrid production, the advantages of using haploids is obvious (Kimber and Riley, 1963; Nei, 1963; Scowcroft, 1975). Normally five or six generations of selfing are required to produce a homozygous line from a genetically heterogeneous population. Inbreeding depression causes inviability or sterility in many of the lines, the cost of which may not be apparent until the third or fourth generation of selfing. The use of the doubled haploid technique automatically selects against any inviable gene combinations and immediately exposes mutations causing sterility. In addition to producing homozygous lines, the doubled haploid technique may have considerable advantage in recurrent selection programs where inheritance is not particulate. Griffing (1975) compared the efficiency of standard recurrent selection methods with those modified by the inclusion of doubled haploid and cloning techniques. With the first of these modifications, individual phenotypic performance of the doubled haploids was evaluated, the population was subjected to truncation selection, and selected individuals were randomly mated to provide the breeding population for the next cycle. The inclusion of cloning techniques provides more precision in evaluating the genotype of a doubled haploid where environmental variance is significant. The comparisons were made where heritability was high (dphenotypic variance was additive genetic), moderate, or low (each separately with environmental variance equal to the additive genetic or with n o environmental variance). The efficiency comparisons showed that genetic gain per cycle of selection is considerably improved by the inclusion of the doubled haploid technique, particularly when total plant numbers are restricted. Given similar cycle lengths the haploid selection procedures can be up to six times as efficient as selection based on diploids. The critical parameter therefore is the relative length of time required per cycle of selection. The use of the doubled haploid technique to increase the efficiency of selection depends solely on the development of rapid doubled haploid extraction procedures. Considerable success has been achieved with tobacco where indeed the value of haploids in breeding programs is being realized. Recent work in the People’s Republic of China has also acheved a substantial improvement in the frequency of haploid production in rice and wheat.

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Lines of tobacco differing in alkaloid content (Collins et al., 1974) and nullisomics for use in genetic analysis have been developed (Mattingly and Collins, 1974). As with many crops, breeding for disease resistance in tobacco is a major objective. Within a relatively short time, compared with conventional procedures, promising disease-resistant lines have been derived from haploids produced by anther culture (Nakamura et al., 1974; Cooperative Group, 1974; Wark, 1977). In yield and quality tests doubled haploid derivatives performed as well as or better than the parental cultivars. Wark (1977 and personal communication) has utilized the doubled haploid technique t o introduce sources of resistance to blue mold (Peronospora tabacina) from related species of Nicotiuna into commercial cultivars. Similarly, resistance to tobacco mosaic virus has been transferred from Nicotiana glutinosa to N. tabacum. Following mutagenesis, haploids have also been screened for resistance to black shank (Phytophthora nicotianae var. nicotianae) (Wark, personal communication). Preliminary tests indicate that some resistant haploids have been obtained. The use of anther-derived haploids in plant improvement appears to have begun in China about 1971. An intensive effort to produce haploids in wheat and rice has led to a substantial increase in the frequency of haploid green plants derived from anther culture. For both wheat and rice the object has been to recover superior haploid segregants primarily from F1 and F2 hybrids. Initial studies on haploid culture in wheat (Ouyang et al., 1973; Wang et al., 1973a) reported a low frequency (11%) of callus formation in cultured anthers and of these less than 30%were capable of regenerating green haploid plants. In 1976 a group from the Institute of Genetics, Academia Sinica (301 Research Group, 1976) reported a dramatic increase in the frequency of wheat haploids by culturing anthers on a medium containing 20%potato water extract, 9%sucrose, 2,4-D (2.0 mg/liter), kinetin (0.5 mg/liter), and iron chelate. When anthers were induced t o form callus on this medium, and then differentiated on Murashige and Skoog medium, the overall frequency of green anther-derived plants was 3-17 times greater than for the controls, the highest frequency being 13.6% of anthers differentiating green plantlets. Initial attempts at haploid culture of rice were successful, but a low frequency (less than 3%) of green plantlets were obtained. Altering the nitrogen source from 10 mM KN03, 12.5 mM NH4N03, and 1.5 mM Ca ( N 0 3 ) 2 , to 3.5 mM (NH4)2 SO4 and 28 mM KN03 (N6 medium) more than doubled the percentage of anthers which produced callus (Chu et al., 1975). Variation in (NH4)2S04 concentration had substantially more effect on callus formation than did variation in KN03 concentration. With the addition of appropriate growth regulators to the N6 medium, a high frequency (75-80%) of plantlet regeneration from callus was found, of which approximately half were albinos. In the two varieties examined in detail the production of anther-derived green plants was 16% and

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12% respectively. Recent work has further indicated that satisfactory induction of plants from anther callus of rice (and wheat) can be achieved without the addition of growth regulations (Chu e t al., 1976). In a recent publication (Yin e t aZ., 1976), a cooperative group of Chinese workers have evaluated a number of lines from anther-derived haploids of rice for agronomic characteristics, disease resistance, yield, etc. Several promising lines are being further evaluated and one line has been named as the variety “Tanfeng” (haploid-derived high-yielding No. 1). The theoretical and practical advantages of the use of haploids in plant improvement are clear. For a given species it is not sufficient merely to demonstrate that haploid plants can be derived from anther culture. This is simply analogous to the occurrence of spontaneous haploids from malfunctions in the process of fertilization and zygote formation. Rather haploidy can only be of value provided haploids can be produced rapidly and in large numbers. An additional limitation may result from competition between pollen-derived embryos during the induction process. Obviously, inviable gene combinations will cause the elimination of many developing embryos and the extent of inviability will depend on the genetic heterogeneity of the breeding population. Competition between developing haploid plantlets must be minimal to ensure that genetic segregation for traits of interest to the plant breeder is fully represented. The ability to culture isolated pollen grains (Nitsch, 1974) largely eliminates this problem. On the basis of realistic assumptions, Nitsch estimated that some 7000 plants could be obtained from a single flower bud of tobacco. This represents immense segregation potential and, for example, could allow the isolation from a heterozygote of an individual carrying up to six recessive alleles. A similar number of F2 segregating genotypes would at best permit the isolation of a homozygous recessive for no more than three loci, which were heterozygous in the parent. Techniques are required to enable this potential to be realized in major agronomic crops. I wish to echo Riley’s (1974) plea that in experiments on anther culture the behavior of the developing gametic sporophyte be monitored closely. In this way principles of wider application may emerge. In this context the correlation of a cytological dimorphism in barley with the propensity t o form pollen callus (Dale, 1975) is noteworthy, as are the earlier observations that ethrel stimulated additional nuclear divisions in pollen grains (Bennett and Hughes, 1972) and that the ribosomal populations of the meiocytes change as they enter meiosis (Mackenzie e l aZ., 1967). I V . Mutant isolation and Selection

The utilization of mutants in understanding biochemical and developmental processes in microorganisms is an obvious paradigm for their potential value in

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plant biology. Moreover, defined mutants greatly facilitate the recognition of rare genetic events such as might result from genetic recombination, mutation, somatic hybridization, and genetic transformation. Apart from these more fundamental uses of biochemical mutants, selecting mutants which cause lesions or alterations in biochemical pathways may be of importance in several aspects of plant improvement. For example, biochemical mutants could be selected for disease resistance, improvement of nutritional quality, adaptation of plants to stress conditions such as occurs in saline soils, elimination of toxins and antimetabolites deleterious to man and animals, and to increase the biosynthesis of plant products used for medicinal or industrial purposes. There are only a few cases where mutants which cause a block in a particular biosynthetic pathway have been recovered in whole plants. These include thiamine-deficient mutants in Arubidopsis (Langridge, 1955) and tomato (Langridge and Brock, 1961), nitrate reductase deficiency in Arubidopsis (OostindierBraaksma and Feenstra, 1973), and a proline auxotroph in maize (Gavazzi et al., 1975). Slightly more success has been achieved in isolating mutants which affect photosynthesis primarily because they affect chloroplast development and can be readily selected (Levine, 1969; Miles and Daniel, 1974; Miles, 1976). Such mutants have been valuable in analyzing basic processes in photosynthesis. The relatively depauperate collection of biochemical mutants in plants probably results from the expense of screening large populations of whole plants for relatively rare mutants. As pointed out by Chaleff and Carlson (1974), the organizational complexity of plants with morphologically and biochemically different, yet interdependent, cells and structures also hinders the isolation of defined biochemical mutants. The ability to manipulate large populations of homogeneous plant cells provides the opportunity to isolate biochemical mutants. Technically it is relatively simple to screen 106-107 cells in culture; screening a similar number of whole plants is very resource-consuming. Because plants can be regenerated from cells of some species the effect of such mutants may be evaluated in mature plants. Dominant and co-dominant mutants can be isolated from diploid, or indeed, polyploid cells. It might appear axiomatic that haploid cell lines would be required to isolate recessive biochemical mutants. However, this might not be the case. Recessive mutants occur in diploid animal cell lines at a frequency considerably greater than would be expected from the frequency of a double mutation event (Terzi, 1974). Recently, Williams (1976) found in the slime mold Dictyostelium discoideum that the frequency of spontaneous mutation to the recessive state at a single locus was only an order of magnitude greater in diploids relative to that in haploids. Indeed, plant cell cultures have been used to successfully isolate biochemical mutants. A discussion of some of these mutants can be found in Chaleff and Carlson (1974, 1975), Widholm (1974b), and Zenk (1974). The only report

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dealing with the recovery of auxotrophic mutants is that of Carlson (1970). In this study he utilized the lethality to growing cells of the incorporation of 5-bromodeoxyuridine as an enrichment procedure for nongrowing auxotrophic mutants. By this procedure Carlson was able to isolate tobacco cell clones which required amino acids, vitamins, or a nucleic acid for growth. These mutants were leaky in that they continued to grow, albeit slowly, on unsupplemented medium. This may have been due to multiple gene copies, for although Carlson (1970) used callus cultures derived from haploids, such haploids do in fact contain two genomes because tobacco is an amphidiploid. It is also possible that plants have alternate biosynthetic pathways. By far the greatest success in isolating biochemical mutants has resulted from selecting mutants resistant to antimetabolites. When nitrate is the sole source of nitrogen for tobacco cells, the inclusion of L-threonine in the medium inhibits cell growth, presumably by blocking the nitrate assimilation pathway (Heimer and Filner, 1970). Under such conditions Heimer and Filner were able to recover a ceIl line which was resistant to the growth inhibitory effects of threonine. The resistance was due to a mutant in the nitrate uptake pathway so that nitrate could be assimilated in the presence of threonine. Mutant cell lines have also been reported which are resistant to the base analogues 5-bromodeoxyuridine (Maliga et aZ., 1973a) and 8-azaguanine (Lescure, 1973; Bright and Northcote, 1975). The BUdR-resistant mutant is controlled by a simple Mendelian gene (Marton and Maliga, 1975). Bright and Northcote (1975) demonstrated that 8-azaguanine resistance resulted from a decrease in hypoxanthine phosphoribosyl-transferase, so lessening the incorporation of the base analogue. Cell lines resistant to the drug streptomycin have been found in Petunia (Binding et aZ., 1970) and tobacco (Maliga et aZ., 1973b). The streptomycin resistance was maternally inherited, and since streptomycin affects the greening of plant tissue it is likely that the mutation occurred in the chloroplasts. It is likely that the chloroplast ribosomes were affected, since streptomycin resistance in bacteria is associated with 70s ribosomes and chloroplast ribosomes are similar to those of bacteria.

A. AMINO ACID ANALOGUE-RESISTANT MUTANTS

Mutants resistant to amino acid analogues are the most thoroughly studied recent biochemical mutants (Widholm, 1974b). Amino acid analogues inhibit the growth of plant cells for several reasons, but the main thrust has been with those that may act as false-feedback inhibitors, i.e., they mimic the natural amino acid in inhibiting one of the enzymes in the biosynthetic pathway. Widholm (1971) obtained circumstantial evidence that tryptophan biosynthesis in cell cultures of several plant species was regulated by feedback inhibition of anthranilate syn-

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thetase. With the tryptophan analogue, 5methyltryptophan, mutants were selected in tobacco (Widholm, 1972a) and carrot (Widholm, 1972b) which were resistant to growth inhibitory concentrations of the analogue. All of the mutants had an altered anthranilate synthetase. Inhibition studies on cell extracts indicated that the enzyme was not inhibited by the analogue, nor indeed by tryptophan, to the same extent as was the enzyme of nonmutant cell cultures. Moreover, an expectation was fulfilled, namely that mutants which lacked feedback regulation would overproduce the specific amino acid. The mutant carrot and tobacco lines had free tryptophan levels 27- and 10-fold higher than normal, respectively. Subsequently, Widholm (1974a) selected an additional 5-methyltryptophan resistant mutant carrot cell line in which the mechanism was due to decreased uptake of the analogue. In this mutant the free tryptophan level was also elevated but the mechanism of how this occurred is unknown. Mutants have also been isolated in carrot and tobacco which are resistant to the phenylalanine analogue, p-fluorophenylalanine (Palmer and Widholm, 1975). The analogue is normally toxic because it is incorporated into protein. The basis for the resistance in the mutants was probably due to decreased incorporation into protein as a result of increased cellular levels of phenylalanine in the carrot mutant, and also presumably for the tobacco mutant, where it appears that the increased phenylalanine was converted to phenolic compounds. The enzyme chorismate mutase from the mutant tobacco cells had reduced sensitivity to inhibition by phenylalanine or its analogue. In the carrot mutant, chorismate mutase was unchanged. The basis for the resistance in the carrot mutant is unknown. A preliminary report (Chaleff and Carlson, 1975) has also indicated that the lysine analogue, S-(P-aminoethyl)-cysteine, which inhibits the growth of rice cells, can be used to select resistant rice cell cultures which have elevated levels of lysine, both in the free amino acid pool and in total amino acids. The levels of other amino acids are also elevated in these mutants. The mechanism of resistance and the basis for the increased synthesis of lysine and other amino acids have not been determined. It would also be of considerable value t o know the lysine content of the grain of plants regenerated from lysine analogue-resistant lines. The isolation of mutants which have elevated levels of certain amino acids is of interest and of possible value to plant improvement because the grains of most crops are deficient in certain amino acids important to human and monogastric nutrition. The limiting amino acid in all cereals is lysine, and maize is also deficient in tryptophan, and wheat and rice are deficient in threonine. Legume grains tend to be deficient in methionine. The principal mechanism which regulates amino acid pools in plants is feedback inhibition, and indeed this has been confirmed by some mutant cell cultures reported previously. Brock et al. (1973) discuss the principles of feedback inhibition in relation to obtaining

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mutants which overproduce lysine. They suggest that the feedback receptor site, presumably on the enzyme aspartokinase, can be inactivated by mutation so that the enzyme is no longer sensitive to feedback inhibition. Such mutants could be selected in the presence of the lysine analogue S-(0-aminoethy1)-cysteine which normally inhibits growth because it mimics lysine in inhibiting the activity of aspartokinase. The cell culture mutants of Charleff and Carlson (1975) tend to support the expectation of Brock et al. (1973). Increasing the level of lysine in the free amino acid pool is of course only the first step. Ideally it is the lysine in the grain storage protein that needs to be increased. The assembly of amino acids into proteins, both catalytically active and storage proteins, is a complex process involving messenger ribonucleic acid (mRNA) synthesis, the coupling of amino acids to transfer RNAs (tRNAs), polypeptide chain initiation, elongation, and termination. As has been pointed out by Brock and Langridge (1975), genetic alterations in the amino acid specificity of tRNAs, which has been done in prokaryotes, could alter the amino acid composition of storage proteins.

B. DISEASE-RESISTANT MUTANTS

The susceptibility of agronomic crops to pathogenic diseases is probably still the major constraint on maximizing yield. The battle against crop pathogens is a continuing one, since pathogenic variants arise by genetic events which render previously resistant crop varieties susceptible. Many bacteria are pathogenic because they secrete toxin lethal to plant cells. The ability t o screen large numbers of plant cells in culture provides a means whereby direct selection for clones resistant to the bacterial toxin could yield resistant genotypes. Mutant clones have been isolated from tobacco cultures which when regenerated into plants have increased resistance to the pathogen Pseudomonas tabaci which causes wildfire disease (Carlson, 1973). The resistance of these plants is not as complete as that in naturally resistant varieties. Methionine sulfoximine was used as the antimetabolite to select the resistant cell clones because it would elicit the same chlorotic response in tobacco as did the bacterial toxin. The relationship between methionine sulfoximine and wildfire toxin is not precisely clear but in bacteria the former interferes with the activity of glutamine synthetase (Brenchley, 1973) and the bacterial toxin is considered to inhibit glutamine synthetase in plants (Sinden and Durbin, 1968). Glutamine synthetase has been shown to be extremely important in cellular metabolism in bacteria (Magasanik et al., 1974), and the same is probable for plants. It is unfortunate that Carlson (1973) did not compare the enzymatic characteristics of the glutamine synthetase in the mutant clones with that in susceptible cells. In 1969, 1970, and 1971 there was an epidemic of southern corn leaf blight (Helminthosponum maydis) because a new pathogenic race arose which attacked

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maize hybrids and inbreds which carried the widely used “Texas” (T) source of male sterile cytoplasm. It has been established that susceptibility t o this pathogenic race was due to a toxin which binds to the mitochondrial membranes of susceptible lines, leading to the uncoupling and inhibition of mitochondrid electron transport (Peterson et al., 1975). Gegenbach and Green (1975) found that the growth of cell cultures derived from maize with T cytoplasm was mhibited by the toxin, whereas the growth of normal cytoplasm cultures was not. Subsequently, they selected a cell clone from the T cytoplasm cultures which was resistant to growth inhibitory concentrations of the toxin. The mitochondria of this resistant clone were no longer sensitive t o the toxin. Resistance was retained when cultures were grown in the absence of the toxin suggesting that the basis of resistance was genetic. Since plants have not been regenerated from this toxin-resistant clone it cannot be established whether the genetic change was nuclear or mitochondrial or whether the cytoplasm was similar t o the parent in respect to male sterility. For any plant disease in which pathogenicity is associated with a toxin, it is relatively inexpensive to treat cell cultures of susceptible, but otherwise desirable, varieties to obtain resistant clones. This would provide an assessment of the chance of recovering resistant mutants by directly exposing the plant population to the toxin. Moreover, if the resistant clones could be regenerated to produce fertile plants, and provided the plant and field resistance correlated well with resistance in cell culture, then plant breeders may have a way of hastening the development of new disease-resistant varieties. The use of tissue culture in this way could provide an alternative t o the expensive and time-consuming conventional method of transferring disease resistance into susceptible but otherwise highly regarded varieties.

C. STRESS-RESISTANT AND OTHER MUTANTS

Plant improvement depends primarily on the evaluation of a phenotype and this of course is a function of many different genetic and biochemical components. However, there is a reductionist approach in biochemistry, plant physiology, and genetics which attempts to provide an elemental description of plant processes in biochemical and genetic terms. As this is achieved, plant tissue culture can be utilized to develop genotypes which have genetic alterations affecting a specific biochemical function. Salinity, particularly in irrigated areas, is a major restriction on realizing yield potential. Also there is an immense crop potential if saline water, and indeed seawater, could be used without the expensive process of desalination. There is a need for salt-tolerant agricultural varieties and this may be achieved by selection since there is a genetic basis to salt tolerance (Dewey, 1960; Abel, 1969; Rush

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and Epstein, 1976). However, where natural genetic variability is absent, tissue culture may provide a solution. It would appear that salt-tolerant clones can be rapidly isolated from plant cell cultures (Nabors et al., 1975; Dix and Street, 1975). These cultures can withstand 4-5 times the salt concentration that inhibits growth of normal cells. Again, the evidence to judge whether plants regenerated from such tolerant clones are also tolerant to high salt concentrations has not yet been provided. Since internal ion concentration is regulated by cellular restriction of ion uptake, or by excretion of adsorbed ion, it is likely that there would be a high correlation between cellular and plant salt tolerance. Crops are often subjected to flooding and it has been postulated that the injury results from the accumulation of alcohol as a consequence of anaerobic respiration in the roots (McManmon and Crawford, 1971). Under anaerobic conditions alcohol dehydrogenase (ADH) catalyzes the reduction of acetaldehyde to alcohol. There are electrophoretic variants of ADH and the “fast” varient apparently is catalytically more active than the “slow” variant (Felder and Scandalios, 1971). Marshall et al. (1973) have shown that maize plants which carry the presumptive catalytically less active form of ADH are more tolerant to flooding. It can be argued that plants deficient for ADH might be even more tolerant to flooding conditions. Indeed a selection system exists for such ADH-deficient genotypes. M y 1 alcohol is converted to the highly toxic acrylaldehyde by ADH (Megnet, 1967), and Schwartz and Osterman (1976) have utilized allyl alcohol as a pollen selection system in maize. Our own research has shown that plant cells are very sensitive to low concentrations of allyl alcohol. We are currently attempting to select allyl alcohol-resistant clones which we predict will be deficient for ADH. This selection system also has the added advantage that ADH can be contraselected, namely, that ADH-mutants will be sensitive to exogenous acetaldehyde which is toxic to plant cells unless metabolized. Using tissue culture cells this selection system may provide a precise genetic means of increasing plant tolerance to flooding. There is no doubt that mutants which are altered in some specific biochemical function can be isolated from cell culture. As has been the case with microorganisms, this will greatly increase the understanding of biochemical processes in plants. The degree to which genetic alterations at the cellular level correlate with altered metabolism in whole plants is as yet largely unknown. The direct value of somatic cell mutants in plant improvement will depend on the extent of t h ~ scorrelation. However, other aspects of genetic manipulation at the cellular level, e.g., somatic hybridization, do require biochemical mutants to provide efficient hybrid selection systems. Although the studies are not specifically related to plant improvement, plant tissue culture is also being evaluated for the production of physiologically active substances, particularly those of medical importance such as steroids and cardiac glycosides (Misawa et al., 1974; Reinhard, 1974). Microorganisms have been

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used extensively for the biosynthesis of commercially valuable metabolites and the efficiency of such biotransformation has been increased by the use of mutants having altered biosynthetic functions. If plant cell cultures prove to be useful for the production of medically important compounds, then assuredly mutants will be selected which yield greater quantities of such compounds.

V.

Plant Cell Protoplasts

A. METHODOLOGY OF ISOLATION

For the purposes of this discussion a protoplast refers t o a cell from which the cell wall has been removed by mechanical or enzymatic methods. It has been possible to isolate protoplasts from plants by mechanical methods, but the yield and quality of the protoplasts is generally low. In 1960 Cocking used a crude enzyme preparation of the fungus Myrothecium yemearia to isolate protoplasts from tomato roots. Since that time, and particularly as a result of the commercial availability of cell wall degrading enzyme complexes (Gamborg and Wetter, 1975), protoplast technology has developed enormously. Several recent reviews examine various aspects of the isolation, culture, and current and proposed uses of plant protoplasts (Cocking, 1972; Tempe, 1973; Eriksson et al., 1974; Gamborg and Wetter, 1975; Vasil, 1976; Gamborg, 1976). Therefore it is not intended to go into specific details or to cite from the extensive literature which is largely covered by these reviews. Only recent and key references will be cited. Protoplasts can be isolated from virtually any plant structure that is not lignified, including leaves, petals, and microsporocytes, and also from plant cell cultures. Leaves have been used extensively for such isolation. To expose the mesophyll cells to the enzyme preparations the epidermis can be physically removed or injured by the use of carborundum (Beier and Bruening, 1975), or leaves can be gently macerated. Enzymatic digestion can be by a sequential process, where mesophyll cells are first released by the action of crude pectinase and the cell walls then degraded by cellulase (Nagata and Takebe, 1970), or by the more common, single-step procedure using an enzyme mixture containing pectinase and cellulase. Since protoplasts are subject to osmotic damage and rupture, an osmotic stabilizer such as mannitol, sorbitol, glucose, or sucrose is required in the culture medium. Factors which affect the quality, quantity, and osmotic stability of isolated protoplasts include the immediate environmental and nutritional history and age of the plants (Shepard and Totten, 1975). Cell suspension cultures are proving extremely valuable for the isolation of protoplasts because of the greater control over the physiological state of the cells and the sterility of the starting material. Cells in early to mid-log phase of growth

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appear to be in the most favorable state for protoplast isolation (Uchimiya and Murashige, 1974). Crude enzyme preparations contain various impurities some of which are probably toxic. While unpurified enzymes can be used with success, an improvement both in yield and in protoplast quality is obtained if the enzyme mixture is cleansed of low molecular weight impurities by passage through a Sephadex G-25 column (Schenk and Hildebrandt, 1969). In our laboratory we have found a substantial improvement in protoplast viability by merely dialyzing the cell wall degrading enzyme mixture overnight in the cold against several changes of distilled water. This appears to remove phenolics, salts, and other low molecular weight impurities. Protoplast preparations have varying degrees of cellular and subcellular debris. Several methods involving repeated sedimentation and resuspension or two-phase liquid partitioning have been used with varying success. We have confirmed a recent report by Larkin (1976) that commercial density buffers containing sodium metrizoate and Ficoll (Lymphoprep, Nyegaard A/S Oslo, Norway; Ficoll-Paque, Pharmacia, Uppsala, Sweden) are excellent for removing debris. Protoplasts as experimental systems per se have already found widespread use in studying virus infection and multiplication (Takebe, 1 9 7 9 , cell organelles and vacuoles (Wagner and Siegelman, 1975), photosynthesis (Nishimura and Akazawa, 1975), the cellular response to toxins from pathogens (Pelcher et al., 1975; Strobel, 1975), and cell wall biosynthesis and deposition (Fowke et al., 1974; Willison and Cocking, 1975).

B. PROTOPLAST CULTURE

Protoplasts are significant to both fundamental and applied genetic research because at least some species can be induced to form a cell wall, divide, and undergo regeneration into plants. This means that genetic modifications that are facilitated using protoplasts may possibly be evaluated in mature plants. Protoplasts can be cultured by embedding in agar medium or suspending in liquid, either as large-volume (25-50 ml) or dropsuspension (about 100 pliters) cultures or as suspensions on agar. The nutritional requirements of cultured protoplasts are similar to those for culturing plant cells but with the addition of osmotic stabilizers (mannitol, sorbitol) and possibly antibiotics (Watts and King, 1973) if bacterial and fungal infection is a problem. The general references previously cited examine the various aspects of the culture and regeneration of protoplasts. Recently, Uchimiya and Murashige (1 976) have systematically examined the nutritional requirements for the recovery of dividing cells from tobacco protoplasts. They found that while growth regulators are not essential for cell wall regeneration, an exogenous auxin is required for

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cell division and a lower than normal sucrose concentration (1.5%) seems optimal. Protoplasts have been successfully cultured in a range of media containing widely varying total salt concentrations (Gamborg, 1976). Uchimiya and Murashige (1976) found Murashige and Skoog’s (1962) basic salts most successful, although they do point out that a systematic approach t o the nutritional requirements for successful protoplast regeneration for a chosen species is most desirable. The time course of cell wall formation and cell division is variable and is treated in detail by Vasil(1976), Willison and Cocking (1975), and Williamson et al. (cited in Gamborg, 1976). The deposition of microfibrils on the plasmalemma membrane begins immediately after removal of the cell wall degrading enzymes, and wall formation can be observed macroscopically, using fluorescent brighteners (Calcofluor), usually within 48 hours. Cell division proceeds thereafter and according to Uchimiya and Murashige (1976), 30% of the protoplasts had r e - f m e d into dividing cells within 5-6 days. The general case is that cell wall formation precedes cell division, but according to Meyer and Abel (1975) division in tobacco protoplasts can occur without rigid cell wall formation. The precise relationship between nuclear division and cytokinesis in plant cells is not known, but at least in Chlamydomonas rheinhardtii, mutants without cell W ~ have been recovered (Hyams and Davies, 1972). A similar mutant in plant cells would be extremely useful particularly if it were a conditional (temperaturesensitive) lesion, whlch under certain conditions could regenerate a cell wall. A mutant clone with a genetic lesion affecting cell wall formation should be relatively easy to select because such a clone would tend to disaggregate in culture. Of course such a mutant might be effectively lethal if cell wall formation is an absolute prerequisite for cell division. The number of species for which plants have been regenerated from protoplasts more or less parallels regeneration studies with normal tissue culture cells. Vasil (1976) and Gamborg (1976) provide lists (and appropriate references) of such species, which include tobacco (Nicotiana tabacum), rapeseed (Brassica napus), asparagus (Asparagus officinalis), carrot (Daucus carota), petunia (Petunia hybrida and P. parodii), tomato (Lycopersicon esculentum), bromegrass (Bromus inermis), Datura innoxia, Ranunculus scleratus, A tropa belladona, and orange (Citrus sinensis). The development of callus from isolated protoplasts has been observed in an additional twenty-odd plant species including soybean (Glycine m a ) , cowpea (Vigna unguiculata), pea (Pisum sativa), sugarcane (Sacchaium sp.), and flax (Linum usitatissimum). Even at this level not one of the major cereal crops is represented. While this list of species might seem impressive, considering that the technology to isolate and culture plant protoplasts has been available for less than a decade, it is unfortunate that no success has been achieved with the cereals and only limited success with the legumes. However, this has not been through a lack of application of protoplast tech-

S

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niques to cereal and legume species. The recalcitrant nature of these species at the protoplast level is a reflection of the difficulties experienced in normal tissue culture studies. Apart from the earlier mentioned uses of protoplasts (Section V, A), as potential cell and plant regeneration systems, protoplasts could facilitate the genetic modification of plant species. There are two broad areas where this might apply. First, they provide a physically amenable system for the uptake of large particles and macromolecules such as DNA, aspects of studies of which will be considered later (Section VI, C). Second, since the rigid cell wall is removed protoplasts can fuse.

C. PROTOPLAST FUSION AND SOMATIC HYBRIDIZATION This topic has been recently and extensively reviewed (Cocking, 1975; Melchers et al., 1975; Vasil, 1976;Gamborg, 1977), and only the broad outlines and recent developments will be presented here. Protoplast fusion does occur spontaneously and this appears to be a consequence of the isolation procedure rather than a result of contact between isolated protoplasts. Fusion between isolated protoplasts can be induced using NaN03, Ca2+ at high pH, and polyethylene glycol (PEG). A comparative evaluation of these methods indicates that PEG-induced fusion is the most effective and reproducible method (Burgess and Fleming, 1974). At a concentration of 20-30% PEG, immediate and extensive protoplast aggregation occurs whch is enhanced by Ca2+enrichment. Fusion is a consequence of the removal of PEG. The relative importance of Ca” in protoplast fusion is also a feature of animal cell fusion, where it has been found that agents which increase the cytoplasmic concentration of Ca2+, e.g., cation ionophores, may enhance fusion (Ahkong et al., 1975). Following protoplast fusion the heterokaryon may form a cell wall and proceed to divide to form callus. Gamborg and co-workers (Gamborg, 1977) have observed division of heterokaryocytes resulting from the fusion of protoplasts obtained from the leaf mesophyll of several species, on the one hand, with protoplasts from cell cultures, primarily soybean, on the other. Nuclear fusion has also been observed in heterokaryocytes of pea and soybean (Constabel et al., 1975a) and carrot and barley (Dudits et al., 1976). For plant improvement, the real value of somatic hybridization lies in the capacity t o transfer genetic information from one species to another. There are now a number of instances where hybrid plants have been recovered following somatic hybridization by protoplast fusion. In each case the recovery of hybrid cell clones depended on the use of a selection system which favored the growth of the hybrid cell. Carlson et al. (1972) recovered a somatic hybrid between two species of tobacco, Nicotiana glauca and N langsdorfii. These two species will

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hybridize sexually, and cells of the tumorous hybrid grow on a media devoid of growth regulators; neither of the two parents grows on such media. T h s provided the basis of Carlson’s selection system and, following the induction of fusion, a number of presumptive somatic hybrid calluses were recovered of which three were analyzed in detail. On morphological, electrophoretic, and chromosome number grounds the somatic hybrid was similar to the sexual hybrid. Fraction I protein analysis of the somatic hybrid revealed that the nuclear-coded small subunits of both parents were present but only the chloroplast-coded large subunit polypeptides of N. glauca (Kung et d., 1975). Carlson’s results have recently been confirmed (Smith et d , 1976) and 23 mature hybrid plants, representing 19 independent fusion events, have been regenerated following PEG-induced protoplast fusion and selection on growthregulator-free medium. Plants from at least 14 of these 19 events were fertile, and corolla, leaf, and plant habit were characteristic of, but somewhat different from, the sexual hybrid. Cytological examination of the 23 hybrid plants revealed a somatic chromosome number of 56-64, which differs from that of Carlson et QZ. (1972) who found a somatic number of 42, which is the chromosome number of the sexual amphiploid representing the 24 from Nicotiana glauca plus 18 from N . lungsdorffi.Smith et QZ. (1 976) explained their results by assuming that successful hybrids resulted from triple fusions with subsequent chromosome loss, which indeed they observed in hybrid plants regenerated from a hybrid callus at different times. Melchers and Labib (1974) recovered somatic hybrids following fusion of protoplasts from two mutant lines of Nicotiana tQbQCUm.These mutant lines carry nonallelic nuclear mutations which affect chlorophyll formation and grow very slowly in strong light. The F1 hybrid between them is normal. Twenty independent hybrids were obtained, and genetic segregation of the two nonallelic mutations in the F2 of the somatic hybrid was similar to that of the sexual hybrid. A similar, although less elegant system, has been used by Gleba et d. (1975) to recover presumptive somatic hybrids also in N. tabacum. Somatic hybrids have also been obtained following fusion of protoplasts from two closely related, sexually compatible species of Petunia (Power et aL, 1976). Hybrid callus was isolated by a selection procedure based on naturally occurring differences between the two species, P. parodii and P. hybrids. On a particular medium P. parodii protoplasts, at best, only produced small (50-cell) colonies and then ceased to grow, whle P. hybrids protoplasts produced viable callus. The complementary part of the selection system was based on the greater sensitivity of P. hybrids protoplasts to actinomycin D. Plants regenerated from selected “hybrid” callus had the expected chromosome number range of 24-28, and flower color and morphology were identical to the sexual hybrid which was distinguishable from either parent. Peroxidase isoenzyme banding patterns differed between the two species. The isoenzyme patterns of the sexual and

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somatic hybrid were identical and represented the summation of the parental patterns. Somatic hybrid plants have been produced from each of four independent experiments.

D . LIMITATIONS TO SOMATIC HYBRIDIZATION

These cases firmly establish that the recovery of hybrid plants by fusion of protoplasts is feasible, albeit this has only been achieved interspecifically and between species that hybridize sexually. In establishing that somatic hybrids could be obtained by protoplast fusion, the availability of the sexual hybrid was an essential benchmark. The examples also confirm the sentiment that a “complementary” genetic system is essential to recover hybrid calluses. In all but one instance (Gleba et ab, 1975) the procedure for isolating the hybrid was designed such that protoplasts and/or cells of both parental species were at a selective disadvantage relative to that of the hybrid cell(s). In each case considerable ingenuity has gone into designing the selection system. The most recent example (Power et al., 1976), where the selection system utilized a naturally occurring difference between species in sensitivity to a particular drug, represents the most generally applicable system used to date. In fact Cocking et al. (1974) have established a considerable spectrum of drug and antimetabolite sensitivities for plant protoplasts. Of course the value of such a system depends on the dominance relationshp which obtains in the hybrid. In a similar attempt at recovering somatic hybrids between soybean and alfalfa, the canavanine resistance of the alfalfa cells was apparently “recessive” to the sensitivity of the soybean because the hybrid clones failed to survive (Constabel et al., 1975b). A more general system of selecting hybrids would involve the isolation of mutants which are resistant to particular antimetabolites. Mutants in which a genetically controlled function has been acquired, such as detoxification of the antimetabolite, is more likely to function effectively in the milieu of a hybrid cell. Such mutants are even more likely to be “dominant” or “co-dominant” if isolated in diploid or polyploid cells. While it is clear that somatic hybridization is possible, the real significance for plant improvement demands that it be possible for species that cannot hybridize sexually. Moreover, it is equally important that genetic information can be transferred as a consequence of somatic hybridization. The next important step is to achieve somatic hybridization and genetic exchange between species which are reproductively isolated. In view of the excitement that the preceding findings have generated, it is germane to consider the potential limitations on the chances of achieving this next step. Apart from temporal and spatial isolation, plant species are reproductively isolated because of gametic incompatibility and/or hybrid breakdown

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(Stebbins, 1966; Grant, 1971). Somatic hybridization should circumvent gametic incompatibility as a barrier to gene exchange. An alternative approach is based on the thesis that gametic incompatibility is a self-nonself immune response and that gametic incompatibility may be mitigated by suppressing this response (Bates and Deyoe, 1973). Hybrid breakdown can occur at any developmental stage following fertilization. One of the genomes may be eliminated as is observed in sexual hybrids between Hordeum vulgare and H. ~ulbosum (Kasha and Kao, 1970). Differential rates of DNA replication could lead to abnormal mitosis and cell division resulting in gross aneuploidy. Abnormal endosperm development can also cause embryo abortion. This latter problem may be overcome by embryo culture, and indeed somatic hybridization would also be expected to succeed where this was the case. Even if somatic hybrids between diverse species were successfully cultured and regenerated into plants, the delicately coordinated process of meiosis and gametogenesis may be insurmountable. Unfavorable gene interactions either between nuclear genomes or between nuclear genes and cytoplasm, and lack of chromosome homology, which will cause abnormal bivalent formation, are widespread causes of hybrid sterility in plants. Obviously, this will eliminate the chance of recovering novel genotypes through segregation. Indeed it is in such a situation where somatic hybridization and somatic cell genetics may provide their real value. Aneuploidy does occur in somatic hybrids (Smith et al., 1976; Power el al., 1976). It is quite conceivable that by repeated somatic “backcrosses” of the hybrid cell to the desirable parent, single chromosome(s) or chromosome segment(s) of the donor parent could be retained. This would of course require a battery of biochemical, genetic, and cytological techniques, some of which are being developed, to identify and retain the specific chromosomes or segments. VI. Genetic Transformation in Plants

Genetic modification in bacteria in addition t o resulting from conjugation, can be effected by transformation with DNA, or transfer can be mediated by episomes or bacteriophages. These are naturally occurring phenomena which have been extensively used experimentally. Moreover, one species of bacterium can be genetically altered by DNA from another species. In eukaryotes there is as yet no known natural alternative to gene transfer, other than by sexual hybridization. However, experimental evidence suggests that nonsexual gene transfer can be achieved with homologous DNA in Drosophila (Fox and Yoon, 1966; Germeraad, 1976) and Neurospora (Mishra and Tatum, 1973); exogenously supplied wild-type RNA can also correct a genetic lesion in Neurospora (Mishra et al., 1975). Cultured mammalian cells have been treated with meta-

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phase chromosomes from a different species, and enzymes specified by the donor can be detected in the treated recipient cells (Burch and McBride, 1975). In plants both observational and experimental evidence indicate that transformationlike phenomena do occur. Most of these studies have involved whole plants, and these will be reviewed briefly because of their possible relevance to the development of novel techniques for genetic modification in plants.

A. MODIFICATION BY HOMOLOGOUS DNA

The treatment of mutant plants with wild-type DNA can be associated with the correction of the lesion at a frequency which is significantly greater than spontaneous correction in the appropriate control plants. The observations which have been reported include the correction of anthocyanin deficiency in petals of Petunia (Hess, 1969), the modification of the effects of the waxy locus in pollen of barley (Turbin er af., 1975), and the modification of genetically determined fruiting characters in Capsicum annuum following DNA treatment (Nawa et al., 1975). This latter observation was similar to graft-induced genetic alteration observed in red pepper (Ohta and van Chuong, 1975). Pandey (1975) has also observed gene transfer following the use of “lulled” irradiated pollen to overcome intraspecific incompatibility in Nicotiana hybrids. Genes which were apparently transferred from the mentor pollen parent modified the incompatibility genotype or the flower color that characterized the maternal parent. Among the many reasons Pandey (1975) advanced to exclude rare pollen mentor nuclei, which might have escaped the very high lethal dose of irradiation, as the cause of such rare genetic events, is that the transformed plants were otherwise phenotypically maternal and distinctively different from the expected hybrid.

B. MODIFICATION BY HETEROLOGOUS DNA

The uptake, integration, and possible transforming ability of foreign DNA has been studied using seedlings, seeds, cultured cells, and protoplasts (Ledoux, 1975; Markham et al., 1975). For some years now Ledoux and co-workers (see Ledoux, 1975) have examined the uptake and integration of bacterial DNA following its application to germinating seedlings. The evidence for the pseudointegration (covalent binding) of the foreign DNA with that of the host DNA is based on the occurrence of DNA, isolated from the treated plants, with a buoyant density intermediate between that of the higher density of the plant DNA and the lower density of the donor bacterial DNA. This intermediate form subsequently could be separated into components which corresponded to the respective buoyant densities of recipient and donor DNA. It has also been

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claimed that a thamine deficiency in Arabidopsis can be corrected with DNA isolated from bacteria prototrophic for thiamine (see Ledoux et al., cited in Ledoux, 1975). However, Ledoux’s buoyant density evidence has not been confirmed. Kleinhofs et al. (1975) using sedimentation analysis were unable to confirm Ledoux’s findings. No intermediate peak was found when axenic plants of pea, tomato, and barley were treated with foreign DNA according to Ledoux’s procedure. Bacterial DNA covalently bound to recipient DNA was found when the plants were not treated axenically. Kleinhofs et al. (1975) argue that the observations of Ledoux are artifactual, resulting from contamination by other bacteria. Lurquin and Hotta (1975) treated Arabidopsis cell cultures with bacterial DNA but were unable t o find any evidence for the intracellular presence of bacterial DNA sequences, either in an integrated or in a free state. With the development of techniques for the characterization of DNA it has emerged that analysis based solely on buoyant density sedimentation is not a sufficient criterion to identify the origin of the DNA. It is essential that base sequence homology be established by DNA hybridization techniques such as can be done on nitrocellulose filters or in solution. The fidelity of base pairing can only be established by thermal renaturation studies. Using these more sensitive techniques, Kleinhofs (cited in Ledoux, 1975) was still unable to demonstrate the presence of donor DNA sequences in the DNA of treated barley seedlings. Therefore, until further evidence is provided, the results of Ledoux claiming integration of bacterial DNA into plant DNA must be viewed cautiously. Plant cell cultures and protoplasts have been used to study the uptake, expression, and possible integration of foreign DNA. Uptake and maintenance of integrity of foreign DNA has been reported in plant protoplasts (Vasil, 1976). In studies such as this, plant nucleases present a real hazard to the integrity of the donor DNA. Moreover, the enzyme preparations used to prepare protoplasts also have considerable nuclease activity (Langridge, personal communication). Nuclease activity, particularly exonuclease, is pH-dependent and activity is substantially impaired at pH 9-10. Another precaution to avoid nuclease activity is to thoroughly wash plant cells or protoplasts prior to treatment. Studies on the expression and possible integration of bacterial DNA in plant cells is at best equivocal. Carlson (1973) infected barley protoplasts with a bacteriophage of Escherichia coli and was able to detect two phage-specific enzymes, S-adenosylmethionine cleaving enzyme and RNA polymerase. The expression of these functions was rapid and transitory. Analogous work with haploid cell cultures of tomato and Arabidopsis suggested that bacterial genetic information for the utilization of lactose as a carbon source could be transferred to plant cells using a transducing phage as vector (Doy et al., 1973). Apparent expression of the transferred genetic information enabled the plant cells to grow on lactose, whereas untreated cells could not, and immunological studies on the

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surviving plant cells indicated that the P-galactosidase was of bacterial, rather than plant, origin. Similar work with the same phage, but with sycamore cells, was initially confirmatory (Johnson et al., 1973). However, subsequent experiments (Smith et al., 1975) have been unable to demonstrate bacterial P-galactosidase. The growth response of the treated sycamore plant cells showed an initial burst of cell division which was not maintained. Smith et al. (1975) doubt that their results provide direct evidence for the uptake and expression of bacterial genetic information. Research in our own laboratory by Dr. V. E. Merriam has also examined the expression of bacterial genes in plant cells, and while some success has been achieved, the results are still equivocal. Tobacco cells, which are sensitive to growth inhibition by the antibiotic kanamycin, were treated with plasmid DNA from Escherichia coli which carried kanamycin resistance. Plant cells were treated under conditions which minimized the degradation of the plasmid by plant nucleases and also included concomitant low levels of irradiation, hopefully to aid integration of the plasmid. Two types of resistant clones were recoveredthose which survived for only a few subcultures in the presence of kanamycin, and those which have been serially transferred many times with no apparent loss of resistance. The majority of these stable clones were derived from experiments where cells were irradiated following exposure to plasmid DNA. Spontaneous kanamycin-resistant mutants have also been recovered. However, in experiments using similar numbers of tobacco cells, the frequency of resistant clones was higher using plasmid DNA than in the controls where plasmid DNA was excluded or replaced by nonplasmid DNA. Similar results have also been reported following treatment of the green alga Chlamydomonasreinhardtii with bacterial plasmid DNA and selection for kanamycin resistance (Gresshoff and Hess, 1977). None of the observations or studies reported in the preceding paragraphs provide unequivocal evidence that foreign DNA can be utilized to genetically modify plants. However, other somewhat more sophisticated evidence indicates that DNA of bacterial origin can be replicated in (Ganem et al., 1976) and translated by (Wang et al., 1976) eukaryote systems and, as mentioned earlier, the stable transfer and expression of genetic information of isolated metaphase chromosomes into mammalian cell cultures of a different species has been achieved. In plants it would seem that the technology has not yet developed to effect the transfer and subsequently detect the presence of foreign DNA. The solution may come from the very recent, and indeed exciting, developments commonly referred to as “genetic engineering.” In the sense of genetic modification by hybridization, selection, and mutation, genetic engineering has been widely practiced by plant breeders. These new techniques however involve the isolation and restructuring of DNA and the reinsertion into a cellular environment where indeed it will function. These techniques are being currently

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explored as a means of generating genetic variation for use in plant improvement. It is appropriate therefore to consider them in this review.

C. MOLECULAR GENE MANIPULATION

The discovery and utilization of two natural phenomena in bacteria have made the in vitro rearrangement of DNA possible. First, circles of double-stranded DNA were found which replicated independently of the bacterial chromosome. These extrachromosomal particles are called plasmids and they endow their host with the capacity to adapt to new environments by conferring properties such as drug or heavy metal resistance, the ability to metabolize long-chain hydrocarbons, and the ability to transfer genetic material by conjugation. Plasmids can also be integrated into the bacterial chromosome. Some plasmids have a wide host range and so genetic information can be transferred interspecifically. Second, was the isolation of a class of bacterial enzymes, restriction endonucleases, which recognize a particular short sequence of DNA and cleave the double-stranded DNA within this sequence. The enzymatic cut leaves singlestranded ends which are complementary. These endonucleases are normally produced for degrading foreign DNA which may happen to enter the cell. Concomitantly, a bacterium can modify its own DNA so that it will not be degraded when nucleases are produced by that cell. Provided the specific sequence is present and unmodified, the restriction nuclease will cut the DNA no matter what its origin, be it prokaryote or eukaryote. Because the singlestrand ends of the staggered cut are complementary, an endonuclease fragment from one species can be annealed with a fragment from another species that has been degraded by the same endonuclease to form a ring which can be covalently closed by incubating in appropriate enzymes (see Cohen, 1975). Hybrid DNA molecules that have been produced to date usually contain a fragment from a bacterial plasmid. This fragment has a very specific function, namely, a replication sequence, or replicon, i.e., a nucleotide sequence to which DNA polymerase can attach so that the hybrid DNA molecule may be replicated. This plasmid usually carries another gene, e.g., drug resistance, so that bacteria which are then transformed by the hybrid molecule can be selected. In this way the hybrid molecule containing the foreign DNA can be multiplied indefinitely and large quantities of it can be purified. Using this procedure of DNA fragmentation by endonuclease and hybridization with plasmid DNA, DNA sequences of Drosophila have been multiplied in Escherichiu coli (Wensink el al., 1974). Some eukaryote DNA sequences specifying a particular function can be isolated, either because of the unique characteristics of that DNA, e.g., ribosomal DNA, or because a specific probe, e.g., mRNA, can be used to isolate the particular gene. These nucleotide sequences

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may also be hybridized with plasmid DNA and multiplied in bacteria. For example, this has been done with the genes of the toad Xenopus laevis that specify ribosomal RNA (Morrow et al., 1974), the histone gene from sea urchin (Kedes et al., 1975), and the gene from rabbit that specifies &globulin synthesis (Rougeon et al., 1975). While the replication sequence of plasmids is probably the most versatile, other such sequences have been used. For example, Struhl et ul. (1976) have hybridized the replication sequence of the bacteriophage h with yeast DNA, and this hybrid molecule multiplies when inserted into bacterial cells. In applying these techniques to transfer specific genetic information t o higher organisms, replication sequences must be found which will permit the hybrid molecule to multiply in the eukaryote cell. The purified DNA of the mammalian virus, SV40, can transform mammalian cells. This has been joined to phage h DNA and the hybrid molecule can be multiplied in monkey cells (Ganem et al., 1976). Because of our interest in developing new techniques for generating genetic variability of potential value to plant improvement, we are currently examining the application of DNA hybridization techniques to plants (Langridge, 1977; Langridge and Scowcroft, 1977). Plant cell and protoplast culture provide a convenient experimental system since it has been established that plant cells, particularly protoplasts, can take up macromolecules such as viral particles and viral RNA which will multiply in the cultured protoplasts. It is more difficult however to find the appropriate vector since the replication sequences that are adapted to multiplication in plants tend to be limited. There are a few DNA molecules which may provide a replication sequence which can be utilized for the purposes of genetic engineering in plants.

I . Possible Molecular Vectors for Gene Transfer in Plants The first of the molecular vectors for gene transfer is the DNA of plant viruses. Most plant viruses are RNA, either single- or double-stranded, and a few have double-stranded DNA. The latter class comprise the caulimoviruses, of which the best characterized one is cauliflower mosaic virus (CaMV) (Shepherd; 1976). Recently, the unrelated potato leafroll virus has also been classified as a doublestranded DNA virus (Sarkar, 1976). CaMV has a molecular weight of 4.7 X lo6 and the purified DNA is infective in plants, but as yet has not been shown to multiply following “infection” of protoplast cultures. Chloroplasts and mitochondrial DNA may also provide a suitable replication sequence. They have respective molecular weights of 90 X lo6 and 40 X l o 6 . Current research is attempting to isolate the replication sequence of the chloroplast DNA. A third possible molecular vector is a plasmid of Agrobacteriurn rurnefaciens. This bacterium is a plant pathogen responsible for a neoplastic disease, crown

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gall. The plasmid of A . tumefuciens may represent the first documented case of a natural example where genetic information is tranferred from bacteria to plants, and in the context of this review warrants further discussion. Infection of wounded plants by A . tumefuciens leads to the transformation of the host cells to the neoplastic, crown gall state. Once transformation has been established the neoplastic growth is self-proliferating in the absence of the inciting bacterium. Ever since this fact was firmly established, considerable effort has been devoted to attempting to understand the molecular nature of the tumor-inducing principle (TIP) (see recent reviews by Drlica and Kado, 1975; Lippincott and Lippincott, 1975). The evidence for a transmissible TIP, although indirect, is compelling. Secondary tumors appear on some infected plant species and these may be free of the inciting bacterium. Bacteria-free crown gall cell cultures can also induce nonself-limiting growth when grafted onto normal, healthy plants. The experimental evidence that bacterial DNA, RNA, or bacteriophage DNA of A . tumefaciens was tumorigenic has been challenged and, indeed, unconfirmed by others (for discussion and appropriate references, see Drlica and Kado, 1975; Lippincott and Lippincott, 1975). Because of the lack of an unequivocal assay for the phenomenon of transformation, most recent studies on the role of bacterial or phage nucleic acids in tumor induction have utilized nucleic acid hybridization techniques. The initial experiments which claimed sequence homologies between tumor DNA and bacterial or phage DNA were based on the hybridization of tumor cell DNA with RNA complementary to A . tumefuciens DNA or PS8 phage DNA. Confirmatory evidence that A. tumefaciens sequences were represented in tumor cell DNA was based on DNA-DNA filter hybridization experiments. However, in these studies the fidelity of base pairing, which can be evaluated by examining the thermal stability of the presumptive DNADNA duplexes, was not determined. When this was done, it was found that no more than 0.02% of the crown gall genome could be homologous with A . tumefuciens DNA. This amounts to less than one bacterial genome per diploid tumor cell. Similarly, little or no homology was found between bacteriophage PS8 DNA and A . tumefuciens DNA. There is now compelling evidence that a large plasmid is involved in the tumor-inducing process. There is a high, though not absolute, correlation between virulence and the possession of a large plasmid (Zaenen er d., 1974). The early demonstration of the transfer of virulence to avirulent Agrobucterium strains by an unknown mechanism of genetic exchange in plunru (Kerr, 1969), now appears to be due to plasmid transfer (van Larabeke er ul., 1974; Watson et ul., 1975). Moreover, many virulent strains lost oncogenicity following growth at high temperatures (Watson er ul., 1975; Bomhoff et ul., 1976) and this was correlated with loss of a large plasmid. Different strains of A. tumefuciens carry different plasmids, and these vary in molecular weight from 96 X lo6 t o I56 X lo6 (Zaenen et uL, 1974). From DNA hybridization studies Matthysse and

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Stump (1976) suggest that approximately 0.1% of bacteria-free tumor cell DNA is A. tumefaciens plasmid DNA. This amounts to approximately 10 plasmids per diploid tumor cell. In addition to virulence there are a number of other properties which now appear to be plasmid linked and they include bacteriophage exclusion (Schell, 1975), sensitivity to a bacteriocin produced by a nonpathogenic species, A. radiobacter (Kerr and Htay, 1974; Schell, 1975), octopine or nopaline synthesis and degradation (Bomhoff et al., 1976). These two unusual arginine derivatives, octopine and nopaline, are normally found in tumorous tissue, and the production of one or other of these compounds is A. turnefaciens strain specific. Moreover, strains with induced octopine in tumor cells are able t o utilize octopine as a source of N for bacterial growth; strains which incude nopaline tumors can utilize nopaline as a source of N. Genetic experiments (Bomhoff et al., 1976) have examined the plasmid-linked nature of octopine or nopaline utilization and systhesis. The plasmid-linked genes have an immense advantage since cells containing this plasmid may be selected and characterized. The crucial question of whether or not the plasmid is integrated into the host cell DNA has not been answered. Neither has it been established that the plasmid per se is capable of causing neoplastic growth of plant cells. Since cultured crown gall cells are growth-regulator independent, and since other biochemical properties are associated with the presumptive tumor-inducing plasmids, it should be possible, using protoplasts, to establish unequivocally whether the isolated plasmid can induce the tumorous state. We have begun research with this specific object in mind.

2. Requirements of the Molecular Vector There are other requirements which we believe essential to utilize such DNA vectors for gene transfer in plants. First, it will be necessary to multiply the hybrid molecules in some convenient organism and at present the bacterium Escherichia coli is most suitable. Therefore the replication sequence that enables the hybrid molecule to multiply in plants must be combined with a replication sequence (i.e., of a bacterial plasmid) permitting multiplication in bacteria. Thus the hybrid molecule could multiply in plants or bacteria. If efficient infection and multiplication of the vectors previously discussed can be achieved in plant cells, as has been obtained for tobacco mosaic virus, then indeed plant cells may provide a convenient milieu in which to adequately multiply the hybrid molecule. The hybrid DNA must aIso carry genetic information that is expressed in both plant and bacterial cells in order to select those few cells which contain the hybrid DNA, As mentioned earlier (Section VII, B), tobacco cells are sensitive to similar concentrations of kanamycin (but not carbenicillin, tetracycline, or

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neomycin) which inhibit protein synthesis in bacteria. No doubt other drugs or antimetabolites will be found that equally inhibit growth of plant and bacterial cells. The A . tumefaciens plasmids have the gene for octopine or nopaline utilization which can be selected for, at least in bacteria. If the induction of growth regulator autotrophy in plant protoplasts can be associated with infection by purified plasmid, then this provides another double selection system. If the hybrid DNA molecule can be taken up, multiplied, and expressed in plant cells, the ultimate object is to integrate i t into the plant genome. As pointed out earlier (Section VI, B) kanamycin resistance appears t o have been stabilized in tobacco cells following transformation by bacterial plasmid DNA. The concomitant use of yirradiation may have led to integration brought about by breakage and reunion. However, this evidence is only circumstantial. 3. Insertion Sequences and Transposons It has generally been believed that integration of DNA into some specific genome can be achieved only as a consequence of recombination between segments of DNA having extensive nucleotide sequence homology or by breakage and reunion. Recently a new class of genetic elements has been characterized in bacteria in which recombination only occurs at the termini of these elements (see review by Cohen, 1976). The nucleotide sequences at these termini are called insertion sequences and they have a defined length of 800-1400 base pairs. There are several different insertion sequences. When such a sequence inserts into a gene the function of that gene is abolished and indeed any other genes distal to the point of insertion relative to the promoter for that operon. When the sequence is excised from a gene the function of that gene, and any other genes in the same operon which were affected, is restored. Any DNA sequence can be contained between two insertion sequences and these termini are arranged so that one is an inverted repeat of the other. Such complexes are known as transposons because they can move from one hereditary element t o another, e.g., from plasmid to plasmid, plasmid to chromosome, or plasmid to bacteriophage, conferring on the receptor element the genetic function contained between the insertion sequences. These transposable genetic elements are probably responsible for the immense genetic diversity of bacterial plasmids, and indeed for the evolution of prokaryote genetic systems. Moreover, the transposable controlling elements that regulate phenotypic expression in maize (McClintock, 1956; Fincham and Sastry, 1974) are consequentially similar to transposons in that they transpose to different chromosomal locations and in so doing influence a variety of genetically controlled functions. These insertion sequences may provide the mechanism for integrating the hybrid molecules into the plant genome. Such a mechanism would mitigate the restraint of the substantial need for sequence homology to effect integration. In the case of the

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association between the plasmid of A. tumefaciens and crown gall, Schell et al. (1977) propose as a working model that insertion sequences are involved in the mechanism of transformation of plant cells to the neoplastic state. If this proposition is substantiated, then molecular genetic engineering in plant improvement is a very real possibility. Provided that a molecular vector can be developed for transferring foreign genetic information into plants, the remaining problem is to decide what genes, or gene-controlled functions, might usefully be incorporated into a plant breeder’s population. A related problem is the identification of the nucleotide sequence for the particular gene in the hybrid molecule, because for the transfer of specific genes it is essential that the hybrid containing the appropriate nucleotide sequence be obtained in quantity. If a bacterium is to be used to multiply the vector, and since eukaryote genes are poorly expressed by prokaryotic protein synthesizing systems, it is probably essential that purified mRNA appropriate for that nucleotide sequence be used as a probe (Kedes et al., 1975; Grunstein and Hogness, 1975). Since such mRNAs are not readily available for plant genes, t h s is unlikely to be of much value. Alternatively a “shotgun” approach can be used where a large number of hybrid molecules, containing random pieces of DNA from the donor genome, are made and inserted into plant cells. The specific hybrid molecule containing the desirable nucleotide sequence is selected by virtue of a property that the gene confers on the recipient plant cell. The specific hybrid selected in plant cells, from among a random set, could then be multiplied in an appropriate bacterium. Even the integration of a vector, containing random pieces of DNA from another plant genome, into a plant species of choice would markedly increase the quantity, and hopefully the quality, of genetic variability on which plant breeders could practice selection. D. PLANT IMPROVEMENT AND DESIRABLE GENES FOR MANIPULATION

In this review, and where experimental information is available, I have presented examples where the asexual transfer of genetic information may be of value to plant improvement (Section IV). These examples have included aspects of disease resistance, tolerance to stress conditions such as salinity or flooding, and the possibility of selecting for amino acid overproducing mutants. Other physiological characters may be subject to genetic modification in cellular systems. For example, freezing injury in plants has long been thought to be due to dehydration injury (Burke et al., 1976), and Towill and Mazur (1976) have recently demonstrated a close correspondence between freezing injury and osmotic injury to cultured plant cells. This would certainly be amenable to cell culture and protoplast studies where resistance to osmotic shrinkage could be selected. The consideration of other possibilities, such as specific gene(s) modifi-

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cation which affect yield, would require the expertise of plant breeders, physiologists, biochemists, and geneticists which is beyond the scope of this review. However, one of the commonly advertised aims of cell culture and genetic engineering is to confer on nonlegume crop plants the ability t o fix atmospheric nitrogen. 1. The Special Case of Nitrogen Fixation

If nonlegumes could be developed w h c h were able to meet even part of their nitrogen requirement directly from biological nitrogen fixation, the benefits from reduced fertilizer use might be enormous. In the best known agricultural nitrogen fixing system, the legume-rhizobia symbiosis, it has been estimated that grain legumes fix as much nitrogen (about 40 X lo6 tons/annum) as is currently provided by the application of chemical nitrogen fertilizers (Hardy and Havelka, 1975). In addition it is estimated that by the turn of the century the demand for nitrogenous fertilizers will rise to 200 X l o 6 tons/annum. There is an obvious need for an alternative. From an examination of known systems for biological nitrogen fixation it is clear that the critical process of reduction of nitrogen to ammonia by the enzyme nitrogenase is restricted to the prokaryotes. There is n o unequivocal example of a eukaryote which synthesizes this enzyme (see Postgate, 1974; Dilworth, 1974). Among the prokaryotes the ability to fix atmospheric nitrogen is fairly widespread, particularly among those classified as primitive. These microorganisms inhabit the rhizosphere and phyllosphere of plants and are found in the water of rice paddies. The contribution of free-living nitrogen-fixing nicroorganisms t o the nitrogen nutrition of plants is poorly understood and probably minimal. Nonpathogenic associations of nitrogen-fixation microorganisms with lower and higher eukaryotes are also widespread in nature, and include the blue-green algal associations with fungi (lichens) and the aquatic fern Azolla (see appropriate chapters in Quispel, 1974). In addition more than a hundred species of nodulated nonlegume plants are known to fix nitrogen; the microsymbiont is almost certainly an actinomycete (Bond, cited in Quispel, 1974). Recently, von Bulow and Dobereiner (1975) have described the natural occurrence of a nitrogen-fixing association between the bacterium Spirillum Zipoferum and roots of monocots. Apart from pasture grass species, this association has been found in roots of maize and sorghum. The extent to which this association provides fixed nitrogen for the host plant is still a matter for conjecture. Clearly then eukaryote plants, during the course of their evolution, have taken advantage of biological nitrogen fixation by entering into associations with microorganisms. Because of its agricultural importance the best understood nitrogen-fixing association is that of the legume-rhizobia symbiosis, where the contribution to the nitrogen nutrition of the host, and t o the nitrogen status of agricultural soils

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is considerable. Recent evidence from our, and other, laboratories has shed additional light on the relative roles of the bacterium and plant in legume symbiosis which indicates that legume symbiosis is not as rigid as previously believed. It was largely accepted that the legume host provided essential genetic information for the synthesis of nitrogenase in legume nodules (Ddworth and Parker, 1969). However, it is now known that at least some species of Rhizobium can fix nitrogen independently of the host (Pagan et al., 1975; McComb et al., 1975; Kurz and LaRue, 1975). Moreover, these rhizobia can also fix nitrogen when cultured with nonlegume plant cells such as tobacco (Scowcroft and Gibson, 1975), wheat, rape, bromegrass (Child, 1975), carrot, and rice (Kurz and LaRue, 1975). It seems therefore that the cellular environment of nonlegumes does not inhibit the process of nitrogen fixation. Studies on the regulation of nitrogen fixation in free-living rhizobia (Scowcroft et al,, 1976; Bergersen and Turner, 1976) indicate that nitrogenase synthesis is not directly regulated by the adenylylation/deadenylylation of glutamine synthetase as has been found for the anaerobic nitrogen fixer Klebsiella pneumoniae (Shanmugam and Valentine, 1975). A further index of the potential flexibility of nitrogen fixation by rhizobia is apparent in its symbiosis with a nonlegume. Trinick (1973) showed that nodules formed on the nonlegume Trema cannabina were due to a slow-growingstrain of Rhizobium. In these nodules, leghemoglobin, which is normally required in legume nodules to regulate the oxygen flux, was not found (Coventry et af., 1976). It is possible that in these Trema nodules an oxypolyphenol oxidase may act as an alternative 02-carrier, to maintain the nodule O2 flux required to support N2 fixation. Alternatively, the rhizobia forming these nodules may have undergone evolutionary adaptation to enable the nodule bacteria to survive and fix N2 without an O2 stabilizing system. This could also account for the fact that only some strains of rhizobia will fix nitrogen under free-living conditions (Pagan et al., 1975). Trinick and Galbraith (1976) have further shown that nodule development in T r e m is more rudimentary than that found in the legumes. They suggest that nitrogen fixation occurs in the extensive infection threadlike structures found in Trema roots as well as in the bacteria-filled host cells. Extensive studies have characterized the genetic regulation of nitrogen fmation in several bacteria, particularly Klebsiella pneumoniae (Brill, 1975). The nif' gene(s) of Klebsiella can be mobilized and transferred to other bacteria using bacterial plasmids. When such information is transferred to the closely related species, Escherichia coli, the nif 'gene is expressed and can be integrated into the E. coli chromosome (Cannon et al., 1974). Using a wide host range plasmid, the Klebsiella nif' gene has also been transferred to nifAzotobacter vinelandii cells (Cannon and Postgate, 1976), and to Rhizobium meliloti and Agrobacterium tumefaciens (Dixon et al., 1976). It was only in the first species that the Klebsiella nif' gene was expressed. In the latter two there

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was no nitrogenase activity but serological tests showed the presence of the molybdoprotein fraction of the Klebsiella nitrogenase in both Rhizobium and Agrobacteriurn. The preceding experimental data make it reasonably probable that all the genetic information for nitrogen fixation is present in, and possibly extractable from, Rhizobium or other nitro-fxing microorganisms. With the development of appropriate molecular vectors, it should not be long before this information can be inserted into other bacteria and even appropriate plant cells. What is not known is whether the intracellular environment of the plant will support active nitrogen fixation; that is, transport gaseous nitrogen, take up and incorporate Mo and Fe, maintain an appropriate oxygen state, provide an appropriate electron donor, and supply energy usable for reduction. In this connection, the evidence that plants have utilized the evolutionary option of developing associations with nitrogen-fixing microorganisms, rather than directly evolving or acquiring a nitrogen-fixing system, is discouraging. On the other hand, the variability of the associations between plants and nitrogen-fixing microorganisms suggests that the deliberate modification of a commensal which inhabits the roots of nonlegumes may indeed be possible. In fact Carlson and Chaleff (1974) have genetically contrived an association between nitrogen-fixing Aztobacter and carrot cells. Here the dependence of the carrot cells on Aztobacter for fixed nitrogen, and the reciprocal dependence of Aztobacter on the carrot cells for an amino acid requirement, led to a relatively stable association. Recent evidence also indicates that infection and subsequent nodulation of legumes by rhizobia may be a function of a plant cell-rhizobia recognition phenomenon involving lectins which are secreted by the legume roots (Wolpert and Albersheim, 1976). It is possible that nonlegume plant cells could be genetically modified t o produce such compounds which in mature plants would interact with rhizobia to initiate infection. The technology to transfer genetic information from one species to another is developing in parallel with a knowledge of the process of nitrogen fixation and in consequence, an appreciation of the real barriers to achieving nitrogen fmation in nonlegumes. It is only through this continuing technology and increasing knowledge that the hope of developing nitrogen fixation for nonlegumes might be fulfilled.

VII. Conclusions

The developments in plant cell culture, allied with the molecular technology of recombining genetic information almost at will, is immensely exciting. However, it would be rash, and indeed a misconstruction, to envisage these research developments as providing a panacea for plant improvement. With the exception of anther culture to produce haploids, none of these developments has yet made

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any direct contribution t o plant improvement. While much of the conceptual groundwork has been done, the utility of somatic cell culture and genetic engineering will depend on an interdisciplinary effort involving research in molecular, cellular, and whole plant biology. The gain from tissue culture will depend on the ease with which economic species can be established in culture and plants can be regenerated from such cell lines. Relevant genetic modification at the cellular level will depend not only on the continual development of basic methodology, but also on the degree to which physiological and agronomic parameters can be defined in cellular and/or biochemical terms. A task force was recently commissioned to make recommendations on how research and development can best be applied to increase food production in an effort t o combat continuing, and sometimes increasing, malnutrition (World Food and Nutrition Study, 1975). They found that for developing countries to meet their rising populations, food production will have to be expanded by 3-4% annually for several decades. Even the highly creditable average annual rate of increase in food production (including yield and acreage increases) of 2.8%during the past 20 years fell short of this goal. The shortfall in demand for food by developing countries was met by imports. Thus, the problem of increasing food production is one not only for the developing countries but also for the more developed exporting countries. Thus, yield increases must be sustained and if possible improved. Yield increases occur through substantial capital investment in land development, machinery, irrigation, fertilizer plants, etc., or through skillful plant improvement. The relative rate at which yield from agricultural crops is increased depends largely on the efficiency of the plant breeder. In this context developments in somatic cell genetics and related areas should pursue one simple objective, namely, to increase the efficiency whereby valuable genetic information can be identified, isolated, and recombined into relevant crop species.

ACKNOWLEDGMENTS

I wish to thank many colleagues from within the Division of Plant Industry, particularly Drs. Janet Pagan and John Langridge, the Australian National University, and from overseas who have at various times offered discussion, ideas, and unpublished manuscripts which have helped formulate some of the sentiments propounded in this review, My gratitude goes to Ms. J. Adamson, Y. Hort, and the Divisional typists for assisting in the preparation of this manuscript.

REFERENCES Abel, G. H. 1969. Crop Sci. 9,697-701. Ahkong, Q. F., Tampion, W., and Lucy, J. A. 1975. Nature (London) 256, 208-209.

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White, P. R. 1942. Annu. Rev. Biochem. 11,615-628. Widholm, J. M. 1971. Physiol. Plant. 25, 75-79. Widholm, J. M. 1972a. Biochim Biophys. Acta 279,48-57. Widholm, J. M. 1972b. Biochim. Biophys. Acta 261,52-58. Widholm, J. M. 1974a. Plant Sci. Lett. 3,323-330. Widholm, J. M. 1974b. I n “Tissue Culture and Plant Science 1974” (H. E. Street, ed.), p. 287-300. Academic Press, New York. Williams, K. L. 1976. Nature (London) 260,785-786. Willison, J. H. M., and Cocking, E. C. 1975. Protoplasma 84, 147-159. Wolpert, J. S., and Albersheim, P. 1976. Biochem. Biophys. Res. Commun. 10,729-737. Woo, S. C., and Su,H. Y. 1975. Bot. Bull. Acad. Sin. 16, 19-24. “World Food and Nutrition Study: Interim Report.” 1975. Nat. Acad. Sci., Washington, D.C. Yeoman, M. M. 1974. In “Tissue Culture and Plant Science 1974” (H. E. Street, ed.), pp. 1-17. Academic Press, New York. Yin, K., Hsu, C., Chu, C., Pi, F., Wang, S., Liu, T., Chu, C., Wang, C., and Sun, C. 1976. Sci. Sin. 19, 227-242. Zaenen, I., van Larebeke, N., Teuchy, H., van Montague, M., and Schell, J. 1974. J. Mol. Biol. 86, 109-127. Zenk, M. H. 1974. In “Haploids in Higher Plants” (K. J. Kasha, ed.), pp. 339-353. Univ. of Guelph, Guelph, Ontario.

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SOIL ORGANIC PHOSPHORUS R. C. Dalal Department of Agronomy and Soil Science, University of New England, Armidale, N.S.W., Australia

I. Introduction .................................................. 11. Organic Phosphorus Content of Soil . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . A. Analytical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Organic Phosphorus Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Organic Phosphorus in Relation to Carbon, Nitrogen, and Sulfur in the Organic Matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Nature of Soil Organic Phosphorus . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . A. Inositol Phosphates . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phospholipids ............................................... C. Nucleic Acids and Their Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . , . . D. Other Phosphate Esters . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . IV. Organic Phosphorus in Soil Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Organic Phosphorus Content . . . . . . . , . . . . . . , . . . . . . . . . . . . . . . . . . B. Nature of Organic Phosphorus . . . . . . . . . . . . , . . . . . . . . . . . . . . . C. Availability of Organic Phosphorus . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . V. Organic Phosphorus Turnover in Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Immobilization of Inorganic Phosphorus into Organic Phosphorus . . . . . B. Mineralization of Organic Phosphorus and Factors Affecting the Process . C. Mineralization of Added Organic Matter . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions ................................................... References ....................................................

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83 84 84 85 85 86 87 88 89 89 90 90 91 92 96 91 100 109 112 113

Introduction

Considerable information is available on the nature and behavior of inorganic phosphorus in soil (Larsen, 1967). The role of organic phosphorus in soil is generally overlooked, mainly because in cultivated mineral soils the greater part of the total phosphorus is in inorganic form. It is therefore assumed that any contribution from organic phosphorus to phosphate uptake by plants is small in temperate regions (Russell, 1973) although it may be significant in the tropics (Williams, 1967). However, soils under pasture may contain 50% (Donald and Williams, 1954) to 84% (Carter, 1958) of the total phosphorus in organic form which, after mineralization, can contribute considerably to the phosphorus nutrition of plants (Halm et al., 1971). Moreover, application of P fertilizers results in the buildup of organic phosphorus in soils under pastures. For example, Rixon (1966) found that 82% to 100% of such fertilizer, added at the 83

84

R. C. DALAL

rate of 172 kg P/ha, was transformed into organic P under irrigated pastures. By using a radiotracer technique, it was observed that 40% of the P applied as superphosphate to the soil under pasture in subtropical environment appeared as organic P within 28 days of application (G. J. Blair, unpublished data). Since organic phosphorus plays a vital role in the phosphorus cycle of pasture soils, this chapter reviews the present state of knowledge of the mechanism of immobilization and mineralization of organic phosphorus in soil; areas of future research are also indicated that may lead to methods of increasing the efficiency of phosphorus utilization by plants. II. Organic Phosphorus Content of Soil

A. ANALYTICAL TECHNIQUES

No direct methods presently exist enabling the determination of the organic phosphorus content of soils. Organic P is determined indirectly by either ignition or extraction methods (Anderson, 1960; Williams et al., 1970). In the ignition method, the organic phosphorus is determined by measuring the differences in the acid-extractable phosphorus in soil samples before and after ignition (Legg and Black, 1955; Saunders and Williams, 1955). The major deficiency of this method arises because the solubility of native soil inorganic phorphorus is increased on ignition; soil organic phosphorus is therefore generally overestimated, especially at higher ignition temperatures (Ipinmidun, 1973). In the extraction method, the organic phosphorus is determined by the difference between the inorganic and total phosphorus in the soil extracts. The most effective extractant for soil organic phosphorus is one that removes the maximum amount of organic phosphorus. This may be achieved either by successive extractions of soil with HC1 and NaOH (Mehta e l aZ., 1954) or by ultrasonic dispersion of soil in NaOH (Steward and Oades, 1972); such extractants unfortunately cause the partial hydrolysis of organic phosphorus. In order to overcome this problem Halstead et al. (1966) suggested acetylacetone as an extractant but it results in low estimates of organic phosphorus (Halstead and Anderson, 1970), possibly because of the complexing effect of heavy metals and clay in soil. Thus Harrap (1963) proposed the use of EDTA to complex the heavy metals whereas Tinsley and Walker (1964) found that hydrofluoric acid and acetylacetone extracted more organic phosphorus than formic acid alone. Recently Tinsley and Ozavasci (1974) used the HC1/HF/TiCl4 solution; most of the inorganic P was dissolved and a major portion of organic P that was precipitated in the soil residue by Ti was extracted with HCl/Cupferron solution and recovered completely by precipitation with Ba from ethanol/ethanolamine solution. The main advantage of these extractants lies in their ability to extract the organic phosphorus mainly in unaltered form.

SOIL ORGANIC PHOSPHORUS

85

B. ORGANIC PHOSPHORUS CONTENT

The organic phosphorus content of soils varies considerably. This fraction may constitute between 20 and 80% of the total phosphorus in the surface layer of soil, although extreme values of 4% of total phosphorus (in a podzolic soil) and 90% (in an alpine humus) have been observed (Williams and Steinbergs, 1958). Kaila (1956) stated that some virgin peat lands may contain as high as 95% of total soil P in organic form, especially a t lower depths. Ipinmidun (1972) found that the parent rock had little effect on the organic phosphorus content of soils derived from gneiss, quartzite, pegmatite, alluvium, sandstone, and iron pan. Soils derived from basalt and basic igneous parent material contain higher amounts of organic phosphorus than those derived from granite, although a lower percentage of their total phosphorus is present as organic phosphorus (Williams and Steinbergs, 1958; Williams er al., 1960). Clay soils are generally higher in organic phosphorus than coarse textured soils but lower than humus soils (Kaila, 1963). Fares et al. (1974) studied the organic phosphorus contents of an andosol, a humic ferruginous podzol, a brown leached soil, a vertisol, a brown calcareous soil, eutrophic mull, and calcic mull. High phosphorus content was associated with high organic matter. An inverse relationship was observed between the degree of polymerization of humic substances and humate organic phosphorus content (Jacquin and Fares, 1974). Organic phosphorus was associated mainly with the fulvic fraction; it decreased in the order: fulvic acids > hurnic acids > humin. Walker and Adams (1959) studied the effect of rainfall on similar parent material. They showed that the phosphorus content of the organic matter decreased as the rainfall and mean temperature increased. Other factors that affect the organic phosphorus content of soils are: drainage [poorly drained soils contain less organic phosphorus than well-drained soils (Williams and Saunders, 1956)] ; soil pH [organic phosphorus content generally increases as the soil pH decreases (Thompson er al., 1954; Kaila, 1963)] ; cultivation [which decreases organic phosphorus content because of greater mineralization of organic matter (Black, 1968)] ; inorganic phosphorus content of the parent material (Walker and Adams, 1958); and sulfur content in areas of low atmospheric returns (Walker and Adams, 1958, 1959).

C. ORGANIC PHOSPHORUS IN RELATION TO CARBON, NITROGEN, AND SULFUR IN THE ORGANIC MATTER

The soil organic matter consists of carbon, oxygen, hydrogen, nitrogen, sulfur, and phosphorus; all the constituents, except phosphorus, are added primarily from the atmosphere. Therefore, the phosphorus in the parent material may govern the accumulation of organic matter in soils (Walker and Adams, 1959); this usually results in phosphorus in the organic matter being present in a fairly

86

R. C.DALAL

fured proportion to nitrogen, carbon, and sulfur. Black and Goring (1953) suggested that the organic matter of mineral soils contains carbon: nitrogen: phosphorus in the ratio of about 110:9:1 (122:10:1.1), but the ratio was wider in organic soils. Taking the organic nitrogen as 10, Barrow (1961) calculated that the average values for organic carbon ranged from 71 to 229, those for organic phosphorus from 0.15 to 3.05, and those for organic sulfur from 1.1 to 1.8. Thus the phosphorus content of soil organic matter is more variable than the carbon, nitrogen, or sulfur contents (Williams and Donald, 1957; Williams er al., 1960). Although this greater variability could have been in part a consequence of limitations of the analytical procedures used, the more variable nature of the phosphorus fraction of the soil organic matter is probably real. This proposition is confirmed by the observation that nearly all the organic phosphorus can be dispersed from a soil comparatively easily, but a proportion of the carbon, nitrogen, and sulfur of the soil organic matter remains undispersed (Russell, 1973). Williams and Steinbergs (1958) suggested that the organic phosphorus could be divided into two parts-the phosphorus intimately bound to the carbon, nitrogen, and sulfur of the humus, and a varying proportion of more or less independent organic phosphorus compounds. Swift and Posner (1 972) observed that the nitrogen and phosphorus contents were greater in the high molecular weight fraction of humic acid. In contrast, the sulfur contents remained constant throughout the molecular weight range. Jacquin and Fares (1974) and Batsula and Krivonosova (1973) found the organic phosphorus mainly in the fulvic acid fraction; only very small amounts were present in humic acids or humin. It is still to be determined how far the nature of organic phosphorus compounds and their combination with organic matter can account for the greater variability in the phosphorus content of the soil organic matter as compared to the carbon, nitrogen, and sulfur contents. The possible cause of this variation-a consequence of soil inositol phosphates which contain neither N nor S , as suggested by Williams (1967), may be only partially correct (Barrow, 1961); inositol phosphates exist in complex forms bound to carbohydrates and proteins (Anderson and Hqnce, 1963; Halstead and Anderson, 1970).

I l l . Nature of Soil Organic Phosphorus

The chemical nature of about half the organic phosphorus in soils is unknown (Anderson and Malcolm, 1974). The compounds so far identified are the inositol phosphates, phospholipids, and nucleic acids (Anderson, 1967). In addition to these three main groups of organic phosphates, there is some evidence that

87

SOIL ORGANIC PHOSPHORUS

phosphoprotein (Anderson, 1967) and sugar phosphates (Omotoso and Wild, 1970; Steward and Tate, 1971; Anderson and Malcolm, 1974) are present in soil organic phosphorus.

A. INOSITOL PHOSPHATES

Among the phosphorus compounds that have been identified so far inositol phosphates certainly predominate (Table I), in some cases accounting for more than 50% of the total organic phosphate present (Anderson, 1967). The parent cyclic polyol (inositol) can have a number of stereoisomers, of which myo-, scyllo-, and D-chiroinositol in the form of phosphate esters have been isolated from soil (Cosgrove, 1966, 1969; Anderson and Malcolm, 1974). Only the myoinositol hexaphosphate isomer has been reported in plants although other inositol isomers may be present in an unphosphorylated form. Cosgrove (1969) has suggested that D -chiro-, scyllo-, and neoinositol hexaphosphates are synthesized by soil microorganisms by a mechanism which does not involve epimerization reactions. However, L'Annunziata (1975) indicated that the soil D-ChirO-, scyllo-, and neoinositol hexaphosphates could be products of microbial TABLE I Distribution of Organic Phosphorus Compounds in Some Soils ~~

Percent of organic phosphorus Soils from

Inositol P

Lipid P

Australia Bangladesh Britain Canada New Zealand Nigeria United States

0.4-38a 9-83 24-58' 11-23f 5-438 23-30: 3-5 2J

0.5-7.0 b 0.6-0.9d 0.9-2.t 0.7-3.1

'Williams and Anderson (1968). bIslam and Ahmed (1973) and Islam and Mandal(1977). 'Anderson (1964). dHance and Anderson (1963). eAnderson (1961). fKowalenko and McKercher (1971b). gMartin and Wicken (1966). 'Baker (1976). iOmotoso and Wild (1970). jCaldwell and Black (1958). kAdams et al. (1954).

-

-

Nucleic acid P -

0.2-2.3b 0.6-2.4e 0.2-1.8k

88

R. C. DALAL

epimerization reactions of the abundant myoinositol or its hexaphosphate from plants or microorganisms. Inositol phosphates in soil exist in complex forms, probably bound in a complex containing carbohydrate and protein. Inositol phosphates may also be linked chemically to larger molecules through their phosphate groups, as in the inositides (Anderson and Hance, 1963). Although the penta- and hexaphosphates of inositol stereoisomers have been shown to be present in soil (Anderson, 1967; McKercher, 1968; Cosgrove, 1966), little is known about the nature of the lower isomers of inositol phosphate. Wild and Oke (1966) found myoinositol monophosphate in CaC12 extracts of soils while Halstead and Anderson (1970) and Anderson and Malcolm (1974) identified the neo-, chiro-, scyllo-, and myoisomers of the inositol containing lower inositol phosphates (di-, tri-, and tetraphosphates). The quantities of lower esters are much less than the penta- and hexaphosphate fraction, possibly because of their lower stability in soil. The ratio of hexa- to penta- is very variable, ranging from over four to one (McKercher and Anderson, 1968). Among the stereoisomers the myo- form is usually the predominant form in soil organic P followed by scyllo-, chiro-, and neo- in decreasing order. However, the cause of the different amounts of these esters present in soil is not known.

B. PHOSPHOLIPIDS

Phospholipid phosphorus content varies from 0.5 to 7.0% of total soil organic P (Table I), with a mean value of 1%(Anderson and Malcolm, 1974). Among the phospholipids known so far (Strickland, 1973), phosphoglycerides possibly form the dominant fraction of the soil phospholipids although there is little information available on the other phospholipids in soil, e.g., phosphoglycolipids, phosphodiollipids, phosphosphingolipids, and phosphonolipids (phospholipids carrying a covalent bond between the phosphorus atom and the carbon of the nitrogenous base). Among the phosphoglycerides, choline phosphoglyceride has been found to be the predominant soil phospholipid (- 40%) followed by ethanolamine phosphoglyceride (- 30%); remaining phospholipids, as determined by current techniques, are present in small amounts (Kowalenko and McKercher, 1971b). The phospholipids in soil may be contributed by plant debris, animal wastes, and microbial biomass. Kowalenko and McKercher (1971a) suggested that a characterization of the fatty acid association with phosphate may be useful in establishing the origin of the phospholipid since bacterial fatty acids tend to be saturated and are either branched or cyclic in form, whereas plant fatty acids tend to be unsaturated and are substituted normally at the 2-position on the choline moiety. They suggested that phospholipids are accumulated in soil from bacterial and fungal biomass. The fact that phospholipids comprise the major

SOIL ORGANIC PHOSPHORUS

89

part of total organic phosphorus in plant tissue (Bieleski, 1973) but only a small amount in soil organic phosphorus (Anderson, 1967) shows that their synthesis and degradation may be fairly rapid in soil. The soil phospholipids may be important in supplying phosphorus to plants; however, this possibility needs to be investigated.

C. NUCLEIC ACIDS A N D THEIR DERIVATIVES

Only a small proportion (up to 3%) of soil organic phosphorus exists as nucleic acids or their derivatives (Table I) in spite of the fact that these are probably added to the soil through decomposing microbial, plant, and animal remains in greater amounts than most other phosphate esters (Anderson, 1967). For example, Bieleski (1973) quoted a typical ratio of DNA:RNA:lipid-P:ester-P of the plant tissue (nonseed portion) as 0.2:2: 1.5: 1 (yrnoles per gram fresh weight). It appears that nucleic acids added to soil are either rapidly degraded or resynthesized and combined with other soil constituents in a form not extracted by existing techniques. The evidence available so far shows that nucleic acids can be rapidly mineralized in soil and incorporated into microbial biomass. Anderson (1961) demonstrated the presence of four nucleic acid bases (adenine, guanine, cytosine, and thymine) in a bound form in humic acid fractions, and from the proportions in which they occurred it was inferred that DNA-derived polynucleotides of microbial origin were present in soil. This was confirmed when two pyrimidine nucleoside diphosphates, thymidine 3‘,5‘-diphosphate and deoxyuridine 3’,5’-diphosphate, were isolated from soil (Anderson, 1970; Anderson and Malcolm, 1974). However, not enough information is available about the RNA-derived polynucleotides in soil (Anderson, 1967). The results of Adams ef al. (1954) and Islam and Ahmed (1973) show that RNA and its derivatives are present in smaller amounts. It is expected that more refined analytical techniques regarding extraction, separation, and identification of the nucleic acid fraction of soil organic phosphorus would assist in understanding the contribution of this fraction to the phosphorus turnover in soil.

D. OTHER PHOSPHATE ESTERS

The three groups of organic phosphates mentioned in the preceding sections account for about half the amount of organic phosphorus in soil and, therefore, much of the soil organic phosphorus is present in as yet unidentified forms. Steward and Tate (1971) isolated phosphorylated compound by gelchromatographic technique from soil organic phosphorus. Electrophoresis revealed that two major components were a nonreducing and a reducing sugar

90

R. C. DALAL

phosphate whose properties suggested that it was a monophosphorylated uronic acid, uronolactone, or a related compound. This compound accounted for 40% of the organic P in 0.1 M NaOH extract. Anderson and Malcolm (1974), from 3 M NaOH soil extracts, detected several monophosphorylated carboxylic acids with C to P ratios of approximately 7 or 8 to 1 and two esters each containing glycerol, myoinositol, chiroinositol, and an unidentified component. It is known that a number of phosphorylated polymers are found in microorganisms (Cosgrove, 1967). It has been suggested that teichoic acids (polymers of ribitol phosphate containing ester-linked alanine), which sometimes comprise up to 50% of cell walls of gram-positivebacteria, may possibly account for some of the unidentified organic phosphorus in soil. Advances in the techniques for extraction, separation, and identification of soil organic phosphorus compounds may reveal hitherto unknown phosphorus compounds which may be contributing significantly to the phosphorus turnover in soil and hence to the phosphorus supply to plants. IV. Organic Phosphorus in Soil Solution

Since the observation of Pierre and Parker (1927) that the amount of organic phosphorus in soil solution may exceed that of inorganic phosphorus, considerable interest has been generated in the amount and nature of organic P in soil solution and its availability to plants.

A. ORGANIC PHOSPHORUS CONTENT

Pierre and Parker (1927) found that the average contents of inorganic and organic P in displaced soil solution of twenty soils were 0.9 and 0.47 ppm P, TABLE I1 Phosphorus in Soil Solution' Organic phosphorus Soil type

Inorganic phosphorus (ppm)

PPm

% of total P

Sand (l)b Sandy loam (5)b Sit loam (8)b

0.02 0.12 0.12

0.28 0.57 0.44

93 83 79

'In extracts obtained by displacement method. Compiled from Pierre and Parker (1927). 0 1927 The Williams & Wilkins Co., Baltimore. bFigures in parentheses are the number of soils examined.

91

SOIL ORGANIC PHOSPHORUS TABLE 111 Effect of Drying on the Concentration of Organic Phosphorus in Soil Solution'

Organic phosphorus

Soil type Broad seriesb (under grass-grazed ley)

Sonning seriesb (under cultivation)

Treatment

Inorganic P (ppm)

ppm

% of total P

Fresh soil Dried at 20°C Dried at 40°C

0.09

0.09

0.14 0.33 1.49

61 69 94

Fresh soil Dried at 20°C Dried at 40°C

0.34 0.44 0.14

0.10 0.11 0.95

23 20 56

0.15

'In 1:2 soil: CaCl, extracts. From Wild and Oke (1966). bTotal soil organic P in Broad series and Sonning series are 620 and 240 ppm, respectively.

respectively; in 1 5 soi1:water extracts the respective values were 0.35 and 0.22 ppm P. Coarse textured soils contained a greater proportion of their solution P in organic form than fine textured soils (Table 11). Fuller and McGeorge (1951) observed that a substantial portion of the total water- and C 0 2 - extractable phosphorus in twenty calcareous soils was present in the organic form. Similarly, Wild (1959) found that the concentration of organic phosphate in CaC12 extracts of soils considerably exceeded that of the inorganic phosphate. The concentration of organic P in soil solution increases considerably upon air drying soil. Thus Wild and Oke (1966) observed that air drying the soil at 40°C increased the proportion of organic P in CaC12 extracts from 61 to 94% in soil under grazed ley, and from 23 to 56% in soil under cultivation (Table 111). The significance of the effect of changes in soil environment due to different cultural practices on the organic P in soil solution should be investigated because of the possibility that it plays a considerable role not only in P movement in soil (Hannapel et aZ., 1964a,b) but also in plant nutrition (Wild and Oke, 1966).

B. NATURE OF ORGANIC PHOSPHORUS

Relatively little information is available on the nature of organic phosphorus in the soil solution. Wild and Oke (1966) identified the myoinositol monophosphate as the major constituent of organic P in the CaCI, extract of soil. Martin (1970) obtained some evidence of phosphate esters in cold water extract of soil, but the other components could not be identified. It appears that a significant proportion of the intracellular organic phosphorus is released into soil solution

92

R. C. DALAL

from the damaged microbial cells with the phosphate ester bond intact. Thus, since organic P in soil solution is not utilized by buckwheat, soybeans, and corn but the plant can absorb P from phytin, lecithin, nucleic acids, nucleotides, and calcium glycerophosphate (Pierre and Parker, 1927), this fact led Rogers et al. (1940) to the conclusion that either organic P does not contain these P compounds in the soil solution or that organic P in the soil solution is present in complex form. A possible explanation is that most of the organic P in the soil solution is actually colloidal in nature and is associated with microbial cells and cellular debris (Hannapel et al., 1964b). The identification of the organic P compounds in soil solution is necessary in order to improve our understanding of their availability and significance in the P nutrition of plants.

C. AVAILABILITY OF ORGANIC PHOSPHORUS

The availability of the organic P compounds that are commonly found in soil (Anderson, 1967) has been demonstrated by many workers. For example, Weissflog and Mengdehl (1 933) showed that, under aseptic conditions, glycerol phosphate, sugar phosphates, inositol hexaphosphate, and nucleic acids were as good a source of P to maize as was inorganic phosphate. Similarly, Rogers et al. (1940) showed that plants can absorb P from inositol hexaphosphate, lecithin, nucleic acids, nucleotides, and calcium glycerophosphate. The availability of inositol hexaphosphate to plants under aseptic conditions has been confirmed subsequently (Szember, 1960; Flaig et al., 1960). Martin and Cartwright (1971) compared the uptake of myoinositol hexaphosphate (IHP) and KH2PO4 labeled with 32P by ryegrass (Lolium perenne). It was found that the availability of added IHP was equal to KH2P04 from low P retention soil but that it was not available to plants when added to high P retention soil (Table IV). One explanation is that IHP was strongly sorbed by high P retention soil. Indeed, Anderson et al. (1974) have shown that IHP was completely sorbed by soil high in P sorption when it was added at the rate of 4 mg P/g soil; the sorption of inorganic P a t that rate was 65% (Table V). Therefore, the low availability of organic phosphorus in soil may be due to the sorption as well as fixation of these compounds by soil colloids and, possibly, by formation of insoluble Fe and Al complexes (Anderson and Arlidge, 1962; Anderson et al., 1974). In spite of the fact that plants can take up P from known organic P compounds, there is no unequivocal evidence that plants utilize organic P from soil solution. Pierre and Parker (1927) observed that organic P in the soil solution was not taken up by plants although inorganic P in the soil solution was almost completely absorbed. However, the results of Wild and Oke (1966) suggest that some of the organic P in the soil solution may be available to plants (Table VI). They showed that the easily hydrolyzable fraction of organic P was taken up

93

SOIL ORGANIC PHOSPHORUS TABLE IV Uptake of Myoinositol Hexaphosphate (IHP) and KH, PO, by Ryegrass' Soil type

Treatmentb

Phosphorus uptakeC (mg/pot)

Coarse sand (low P retention)

Control IHP KH, PO,

1.23k 6.691 6.131

Lateritic podzolic (high P retention)

Control IHP KH, PO.,

0.08m 0.04m 2.6511

'Adapted from Martin and Cartwright (1971), by courtesy of Marcel Dekker, Inc., New York. bLabeled IHP and KH,PO, were applied at 114 mg P/pot (approximately 38 mg P/kg soil). 'Means followed by letters not in common differ significantly at P < 0.01.

readily by clover but that the fraction resistant to hydrolysis had a low availability to plants grown under aseptic conditions. Moreover, organic phosphates forming water-soluble complexes with Fe and A1 (organometallic phosphates) can be utilized by plants (Sinha, 1972). Another possibility that organic phosphorus in the soil solution may be important to P nutrition of plants is that phosphatase enzymes, excreted by the plant roots could hydrolyze this fraction thus releasing inorganic P. That the plant roots possess phosphatase activity has been confirmed (Ridge and Rovira, 1971; Martin, 1973). Moreover, it has been shown that P-deficiency in plants increases phosphatase activity (Table VII). In addition, microorganisms present in the soil may also be involved in hydrolyzing organic compounds. For example, Cosgrove (1970) isolated from a soil an organism possessing a high TABLE V Sorption of Inositol Hexaphosphate (IHP) and Inorganic Phosphorus (Pi) by Two Soils' Phosphorus sorbed (%) P added (mg/g soil) Soil type

4

10

20

Sand (low P retention)

IHP Pi

7 12

1 7

1 4

Basic igneous (high P retention)

IHP Pi

100 65

25 32

9 22

'Calculated from Anderson et al. (1 974). In 0.5 M acetate buffer at pH 6 .

94

R. C. DALAL TABLE VI Phosphorus Availability to Clover of the Three Fractions of the Soluble OIganic P in Broad Series‘

Fractionb

Percent available’

78 31 93 ‘Taken from Wild and Oke (1966). I bThree fractions accounted for 3 0 4 0 % of organic P in soil solution. Myoinositol monophosphate was the dominant constituent in P, and P, and probably in P, fractions. ‘Compared with availability of inorganic phosphate taken as 100.

activity of the specific enzyme inositol hexaphosphate phosphohydrolase. Greaves and Webley (1965) found that 3 0 3 0 % of bacterial isolates from the rhizosphere of pasture grasses possessed phytase activity; however it is doubtful whether the occurrence of bacteria possessing the phytase activity in the rhizosphere will increase the dephosphorylation of myoinositol hexaphosphate at the root surface above the activity due to plant enzymes (Martin, 1973). Further, it is uncertain whether phosphatase activity in the presence of low concentration of phosphate esters in the solution has significance (Bieleski, 1973). Recently there has been a considerable interest in the possible increase in availability of organic phosphorus to plants resulting from the infection of plant roots by mycorrhizae. Paterson and Bowen (1968, cited in Bowen, 1973) showed that ectomycorrhizal fungi in culture could use sugar phosphates, nucleotides, and inositol hexaphosphate as sources of energy and phosphate and that mycorrhizae of P. radiata exhibited surface phosphatase activity. The phosphatase activity of mycorrhizal and nonmycorrhizal roots are compared in Table VIII. Since mycorrhizal association occurs commonly even in cultivated TABLE VII Phosphatase Activity of the Roots of Spirodeta oligorrhiza under Phosphorus Sufficiency and Deficiency Conditions‘

Treatment

Enzyme activity (ex.) b (~10-3)

Control P deficiency (1 1 days) P deficiency (14 days)

64 1 139

15

‘Calculated from Bieleski and Johnson (1972). bl e.u. hydrolyzes 1 ,mole p-nitrophenyl phosphate per minute per gram fresh weight at 25°C.

95

SOIL ORGANIC PHOSPHORUS TABLE VIII Phosphatase Activity of Mycorrhizal and Nonmycorrhizal Rootsu Enzyme activityb Date of sampling

Mycorrhizal Roots

Nonm ycorrhizal

19/2/73 24/3/73 1014173

3.90 5.70 5.40

1.05 0.68 2.25

‘From Williamson and Alexander (1 975). bMicromoles X of p-nitrophenyl phosphate hydrolyzed/mm2 root surface h-’ .

plants (Strezemska, 1974), it may be of considerable importance in P nutrition of plants. The advantage of mycorrhizal association in the use of organic phosphorus is the ability of mycelia to penetrate soil pores and soil organic matter at distances away from the root, thus exploiting a greater soil volume than uninfected plants as well as competing positively with other soil microorganisms. In that way mycorrhizal-infected plants can absorb greater amounts of phosphorus. Further, since certain mycorrhizal fungi can grow at low water potentials, when other organisms are senescing and releasing organic phosphates, it would be of considerable advantage to the mycorrhizal-infected plants (Bowen, 1973) in dephosphorylating, absorbing, and translocating the absorbed P. However, Hayman and Mosse (1972) observed that the plant roots could not utilize organic phosphate even in the presence of vesicular-arbuscular mycorrhiza. They concluded that the main role of mycorrhiza was the provision of extra nutrientabsorbing surface. Because of these conflicting reports, it may be useful to investigate the significance of organic P t o mycorrhizal-infected plants. In summary, it can be concluded that: (a) the concentration of organic phosphorus in soil solution exceeds that of organic phosphate, (b) the hydrolyzable soluble organic phosphate can be utilized by plants, and therefore it is necessary to characterize the organic phosphorus compounds in soil solution, and (c) mycorrhizae may increase the availability of organic phosphate by producing dephosphorylating enzymes. It is necessary to determine whether the soluble organic phosphorus can be replenished when its concentration is reduced by plant uptake. Moreover, the environmental factors that govern not only the concentration of soluble organic P but also its turnover (in whole or in part) should be studied. Since the organic phosphorus in the soil solution is more mobile than the inorganic phosphorus [and indeed in calcareous soils, Hannapel et al. (1964a,b) showed that 95% phosphorus movement in the soil is in organic form], it would be of interest to investigate this phenomenon especially in soils where organic phosphorus is of the predominant form in the soil solution.

96

R. C. DALAL V. Organic Phosphorus Turnover in Soil

Since organic phosphorus is a part of soil organic matter, it tends to follow the pattern of accumulation and loss of organic matter as a whole. The process of buildup of organic phosphorus may be termed immobilization, i.e., available inorganic phosphorus is converted biologically into organic phosphorus compounds which are unavailable to plants. The microbial conversion of soil organic phosphorus into inorganic phosphorus is termed mineralization. The immobilization and mineralization of phosphorus can occur concurrently in soil. Halm et al. (1971) studied the phosphorus cycle in a native grassland system and presented it diagrammatically in Fig. 1. Examination of Fig. 1 shows that the large concentration of phosphorus is in the soil fauna and microorganisms, and the organic and inorganic fractions. The rate at which these fractions are made available to the soil solution controls the phosphorus supply. The amount of phosphorus in the birds, grasshoppers, small mammals, and other inverteorates (consumers), and in the above-ground plant material, at any given period is very small when compared to the extremely large amount tied up in the organic and inorganic phosphorus fraction of the soil. It is also interesting to note that soil fauna and microorganisms together contain more phosphorus than t!ie total amount in the plant material. The phosphorus cycle of a native grassland ecosystem is summarized as follows (Halm et al., 1971). The phosphorus in litter is attacked by fungi and is physically moved into the soil in fungal hyphae which are then attacked by

FIG. 1. Phosphorus cycle in a native grassland system (in parentheses is P expressed in kg per hectare per 30 cm soil depth). Adapted from Halm ef al. (1971).

SOIL ORGANIC PHOSPHORUS

97

bacteria providing a continuing source of organic phosphorus (Clark and Paul, 1970). The more soluble fraction of this phosphorus is immobilized by new microbial tissue or converted into more resistant compounds forming soil humus. On mineralization, it goes into soil solution where it may be taken up by plants, adsorbed by soil colloids, and fixed into unavailable inorganic form or again appropriated by microorganisms. Thus both processes, immobilization of inorganic P and mineralization of organic P, occur simultaneously in the soil and only the difference in the rates of immobilization and mineralization of organic P can be observed at any given time. A. IMMOBILIZATION OF INORGANIC PHOSPHORUS INTO ORGANIC PHOSPHORUS

The available literature on phosphorus turnover in soil reflects that more studies have been carried out on the factors which govern phosphorus mineralization than on organic phosphorus buildup in soil. Considerable amount of native inorganic phosphorus has been transformed into soil organic phosphorus over the years (Walker and Adams, 1958). Since carbon, nitrogen, sulfur, and phosphorus are associated in fairly definite proprotions in soil organic matter, a deficiency of either sulfur or phosphorus may limit nitrogen fixation by legumes or microorganisms. In areas where sufficient sulfur is supplied from the atmosphere, the organic matter buildup and hence organic phosphorus accumulation of soil would be determined by phosphorus content of the parent material. Indeed, Walker and Adams (1958) observed that the phosphorus content of the parent material was a major factor governing the accumulation of organic phosphorus in soil. Subsequently a number of workers have observed a close relationship between organic P and total phosphorus content of soil (Kaila, 1963; Syers and Walker, 1969; Walker and Syers, 1976). In soils where native inorganic phosphorus is low, as in many Australian soils (Jackson, 1966), the application of inorganic phosphorus, especially to legumegrass pastures, should result in an organic phosphorus buildup. Donald and Williams (1954) found that the application of superphosphate to subterranean clover (Trifolium subterraneum L.) grown in podzolic soils for 26 years resulted in an increase in organic P from 53 to 9 6 pprn (increase of 4 ppm organic P per 9.5 kg of P applied). The results of Jackman (1955) and Rixon (1966) (Table IX) show that organic phosphorus buildup can be fairly rapid under favorable conditions although the rate of accumulation would be different depending upon a number of environmental, soil, and plant factors. Factors other than inorganic phosphorus supply may limit organic phosphorus accumulation in soil. Williams and Donald (1957) suggested that the rate of organic P accumulation under legume pastures may be limited by the insufficient

98

R. C. DALAL TABLE IX Increase in Soil Organic Phosphorusunder Irrigated Pastures" Increase

in organic P by year 5 Pasture

Organic P by year 1 (ppm)

ppm

% '

Annuals Wimmera ryegrass (Lolium rigidum Gaud.) Subterranean clover (Trifolium subterraneum L.)

129 136

26 82

22 60

Perennials Perennial ryegrass (L. perenne) White clover (T.repens L.)

140 157

75 83

54 53

'Calculated from Rixon (1966). Superphosphate added, at the rate of approximately 50 kg P per hectare per year, in the years 2, 3, and 4.

amounts of sulfur supplied in the superphosphate. The results of the experiments conducted by McLachlan and Norman (1962) did not support their suggestion, however; they indicated that phosphorus rather than sulfur could be the limiting factor in organic phosphorus buildup. Jackson (1966) has indicated the situations in pastures where phosphorus or sulfur could be limiting for organic matter and hence organic phosphorus accumulation. The addition of soil carbohydrate or organic material having a large carbon to phosphorus ratio leads to increased microbial activity and the formation of organic phosphorus from a pool of inorganic phosphorus in soil (Cosgrove, 1967). Van Diest (1968) noted accumulation of soil organic phosphorus in Coastal Plain Soils of New Jersey owing to the addition of fertilizer P and energy materials through crop residues. Ghoshal and Jansson (1975) have elegantly demonstrated the immobilization of inorganic phosphorus when glucose-C as the energy source was added (Table X). The organic carbon:organic phosphorus ratio in soil and plant residues added to soil has been used to predict net immobilization and mineralization of phosphorus in soil (Black and Goring, 1953; Thompson et a l , 1954; Alexander, 1961; Tisdale and Nelson, 1975). It has been suggested that if the organic carbon:organic P ratio is 200: 1 or less, mineralization of phosphorus occurs and if the ratios are 300:l or more than immobilization occurs. Thus the critical level of phosphorus in organic material which serves as a balance between immobilization and mineralization is about 0.2%(Alexander, 1961). However, Enwezor (1967) observed that organic C:organic P ratio in the soil was an unreliable index for predicting immobilization or mineralization. Birch (1961) suggested that the cause of the inconsistency of the C:P ratio as an index of

99

SOIL ORGANIC PHOSPHORUS TABLE X Immobilization of Added Phosphorus as Affected by Phosphorus and Glucose-Carbon' Phosphorus added (ppm) 33.3 33.3 166.6 166.6 166.6

Glucose-C added (%)

P immobilizedb (ppm)

-

19.3 22.3 19.4 42.8 46.2

0.25 -

0.25 0.50

'From Ghoshal and Jansson (1975). bIncubated for 15 days.

immobilization or mineralization of P may be due to the variable but significant amounts of inorganic phosphate present in the organic materials. Further, Barrow (1960) suggested that this inconsistency could be a consequence of the suboptimal supply of nitrogen and/or sulfur in the soil; in either case the formation of soil organic matter would be inhibited. The addition of N through either fertilizers or atmospheric fixation by legumes could result in the immobilization of inorganic phosphorus. However, it needs to be confirmed by labeling the phosphate pool with 33Pin the added organic matter and then following its decomposition in relation to mineralization and immobilization processes. Soil organic phosphorus accumulation is also governed by environmental factors other than the nutrient supply. Organic phosphorus accumulation decreases with increase in leaching as a result of rainfall (Walker and Adams, 1959). Higher temperatures cause greater mineralization and hence less net immobilization although suboptimal temperature also decreases immobilization rate because of reduced biological activity. Suboptimal moisture has a similar effect. Type of vegetation also influences organic phosphorus accumulation in soil. Ipinmidun (1972) found that soils from southern Guinea savanna had a higher organic phosphorus content than those from the Sudan vegetation zone. Soils under forest immobilize higher amounts of phosphorus than those under grass (Enwezor and Moore, 1966). Rixon (1966) observed that organic P accumulation was significantly lower under Wimmera ryegrass than under clover or perennial ryegrass (Table IX). Increase in soil acidity could restrict organic P accumulation. Thus Simpson et aZ. (1974) observed that organic P remained unaffected by the 9-fold increase in superphosphate application from 64 kg P/ha to 579 kg P/ha; the possible increase in acidity due to N fertilizers appeared to be the cause. The mechanism of organic phosphorus accumulation in soil has not been completely worked out. Van Diest (1968) suggested that increases in soil organic P might be induced either by increase in the soil microflora following application

100

R. C. DALAL

of fertilizer P, or by accumulation of crop residues containing a fraction of organic P resistant to rapid hydrolysis, or by a combination of these two processes. Evidence for both the processes exists in the literature. Birch (1961) found no pronounced mineralization of plant organic P (incubated with sand) during decomposition periods up to 3 months. In soil, protection from decomposition might be even stronger due to possible sorption of organic phosphorus compounds on clay minerals (Pinck and Allison, 1951; Goring and Bartholomew, 1950, 1952) and hydrated oxides of iron and aluminum (Anderson and Arlidge, 1962). Such sorption decreases the rate of mineralization by enzymes (Greaves and Webley, 1969). In fact, Anderson ef al. (1974) showed that the stability of phosphate esters was due to their sorption by soil, and possibly the formation of insoluble Fe and Al complexes with esters. Since phosphorus accumulation in the soil organic matter follows the organic C, N, and organic S accumulation in soil (Jackman, 1964), organic phosphorus is almost certainly accumulated in soil as the result of microbial activity. For example, before 1963, soil phytin (metal salts of inositol hexaphosphate) was assumed to be entirely of plant origin (Birch, 1961). However, Cosgrove (1963, 1966) and others (L'Annunziata, 1975) have shown that phosphate esters of D -chiro-, scyllo-, and neoinositol are synthesized by microorganisms in soil (Section 111, A). Although the origin of soil phospholipids is not known (Cosgrove, 1967), it has been suggested that these are accumulated in soil from bacterial and fungal biomass (Section 111, B) whereas nucleic acid phosphorus in soil has been found to be of bacterial origin (Anderson, 1967). As shown earlier (Table X), organic phosphorus can be synthesized by microflora from inorganic phosphorus and available organic C. However, long-term studies by Halstead and Sowden (1968), where organic amendments to a sand and a clay soil were added, gave no evidence to either support or deny the assumption that accumulation of organic phosphorus was caused by microbial rather than by plant residue effects. It is hoped that the better understanding of soil organic phosphorus through improved techniques such as ion exchange and gel-chromatography , nuclear magnetic resonance and mass spectrometry, as suggested by Halstead and Mc Kercher (1975), would assist in elucidating the mechanism of organic phosphorus buildup and its possible relationship to other constituents in soil organic matter (organic C, N, and S), and their subsequent (and possibly concurrent) mineralization in soil.

B. MINERALIZATION O F ORGANIC PHOSPHORUS AND FACTORS AFFECTING THE PROCESS

Soil organic phosphorus contributes to the phosphorus nutrition of plants primarily after being mineralized into inorganic phosphorus. It has been shown in the laboratory that in incubated soil, organic phosphorus decreases with

101

SOIL ORGANIC PHOSPHORUS TABLE XI Changes in Organic and Inorganic Phosphorus upon Cropping' Percent change from virgin soil

Manured Unmanured

Organic P

Inorganic P

-3 5 -3 I

+29 + 2

'Estimated from Haas el a[., Soil Science Society of America Proceedings, 25, 214-218 (1961). The period of cropping ranged from 30 to 48 years at the different locations in the Great Plains region of the United States.

similar increase in extractable inorganic phosphorus (Thompson et al., 1954; Van Diest and Black, 1959). The decrease in organic phosphorus when virgin soils are brought under cultivation is possibly due to mineralization, for Haas et al. (1961) observed that the decrease in phosphorus from the soil on cropping was approximately equal to the loss of organic phosphorus (Table XI). Further evidence that soil organic phosphorus could be mineralized is that phosphatase activity is directly related to organic phosphorus content (Gavrilova et al., 1974). Another form of evidence for organic P mineralization is provided by analogy with the organic nitrogen and carbon mineralization. Thompson et al. (1 954) showed that the rate of organic phosphorus mineralization was positively correlated with the rates of nitrogen and carbon mineralization. The amounts of organic phosphorus, nitrogen, and carbon mineralized were proportional to these constituents present in the soil organic matter, both in virgin and in cultivated soils (Table MI),although this relationship does not always exist (Williams and Lipsett, 1961). The mineralization of organic phosphorus in soil is largely due to the combined activities of the soil microorganisms and the free enzymes, phosphatases TABLE XI1 Regression of Mineralized Nitrogen and Organic Carbon on Mineralized Organic Phosphorus in Virgin and Cultivated Soilsa Soil

Regression coefficient Constant term Significant at P =

Virgin soils Cultivated soils

2.06 2.6 2

81 52

0.01 0.01

Organic carbon Virgin soils Cultivated soils

20.6 1 28.15

829 515

0.01 0.01

Nitrogen

aTaken from Thompson et al. (1954). 63 1954 The Williams & Wilkins Co., Baltimore. The regression equation is: N (or C) = a P + b, where a is regression coefficient (which did not significantly differ between virgin and cultivated soils) and b is a constant term (which differed significantly between virgin and cultivated soils).

102

R.C. DALAL

(exo- as well as intracellular phosphatases released following the lysis of microbial cells), present in soil. The factors that regulate the activity of microorganisms thus mainly govern the mineralization of organic phosphorus in soil.

1. Temperature Since the optimum temperature for growth of most bacteria is between 30" and 45°C (Stanier et at., 1971), mineralization of organic phosphorus increases with increasing temperature, particularly above 30°C (Thompson and Black, 1949; Van Diest and Black, 1959; Acquaye, 1963). Eid et al. (1951) observed that the optimum temperature for soil organic phosphorus mineralization was 35°C. Thus Floate (1970) did not observe phosphorus mineralization from plant material below 30°C;in fact net immobilization of phosphorus occurred at such temperatures (Table XIII). Similarly, Eid et al. (1951) showed that organic phosphorus was not available to plants at 20°C. Saunders and Metson (1971) and Dormaar (1972) observed that the soil organic P increased during winter and decreased during spring. From these observations, Williams (1967) concluded that in tropical soils, mineralization of organic phosphorus may contribute significantly to the phosphorus nutrition of plants, whereas in temperate soils the contribution of organic phosphorus may be much smaller. In fact, Williams and Lipsett (1961) found that only about 15 kg P per hectare or 17% of the total organic P was mineralized during 50-60 years of wheat cultivation in (temperate) New South Wales. Another consequence of the effect on P mineralization is that in temperate soils organic phosphorus is mineralized TABLE XI11 Effect of Temperature and Moisture on Mineralization of Phosphorus from Plant Material and Sheep Feces Incubated for 12 Weeks' Percentage of original P mineralized Temperature ("C) WHC at 10°C (%) Material incubated Plant material Ab Bb Feces AC

BC

Pcontent

C/P ratio

N/Pratio

5

10

30

25

50

100

0.135 0.110

303 364

10.3 8.1

-49 -34

-29 -24

-5 6

-10 3

-14 - 2

-12 -11

0.137 0.628

63 64

2.8 2.4

-13 11

2 3

7 13

10 3

8 0

2 2

'Adapted from Floate (1970). bA and B plant materials were Festuca-Agrostis and Nardus type, respectively. 'Feces from sheep fed with A and B type of plant material, respectively.

SOIL ORGANIC PHOSPHORUS

103

more slowly than carbon, nitrogen, or sulfur when the soil is cultivated, causing an increase in the proportion of organic phosphorus in soil organic matter (Thompson et al., 1954; Williams and Lipsett, 1961); in contrast it is mineralized at approximately the same rate as carbon and nitrogen in tropical soils (Thompson et al., 1954; Paul, 1954). The knowledge of the chemical nature of organic phosphorus in such soils should enable the processes involved in the mineralization of soil organic phosphorus to be elucidated.

2. Moisture Adequate moisture is essential for mineralization of organic phosphorus from soil organic matter and decomposing plant residues. Feher et ~ l (1939) . found that citric acid-soluble phosphorus (organic phosphorus) in soils was greater after incubation at 50 to 75% of the water-holding capacity (WHC) than at either 25 or 90% WHC. Floate (1970) observed that the mineralization of organic phosphorus from plant material and sheep feces was enhanced at 25% WHC compared with that at 50 or 100%WHC (Table XIII). Considerable mineralization of inositol hexaphosphate has been found to occur when soils are submerged (Furukawa and Kawaguchi, 1969; Islam and Ahmed, 1973). Mineralization of organic P in laboratory experiments was found to be greater in flooded soils than in soils maintained at field capacity (Campbell and Racz, 1975). It was suggested that this might have been due to anaerobic conditions occurring in flooded soils. Evidently information about the effects of moisture on the mineralization of phosphorus from soil organic matter or plant materials is too scarce to draw any firm conclusions; more work, therefore, is needed to evaluate the effects of moisture on organic phosphorus mineralization. Further, some of the reported results on the effect of moisture on soil organic P are confounded with the effect of aeration. Alternate wetting and drying of soil enhances phosphorus mineralization. For example, Birch and Friend (1961) found that the phosphorus in an organic soil was completely mineralized as a result of 204 wetting-incubationarying cycles. This was in marked contrast to organic carbon and nitrogen; 37% of organic carbon and 54% of nitrogen remained in organic form after such cycles. The mechanism of the alternate wetting and drying effect has not been established, but it has been suggested that an important part of the organic matter which decomposes during the drying process is the one that disperses into the soil solution on wetting (Skyring and Thompson, 1966; Russell, 1973). It is also possible that since wetting and drying breaks up water-stable soil aggregates, the humic matter that has been inaccessible to the soil microorganisms becomes exposed for decomposition, for Soulides and Allison (1961) showed that multiple wetting and drying cycles caused a greater reduction in water-stable aggregates than did a single cycle. The differential effect of wetting and drying cycles

104

R. C. DALAL

on various components (such as carbon, nitrogen, and phosphorus) of the soil organic matter needs to be investigated (Birch and Friend, 1961).

3. Aeration The effect of aeration on phosphorus mineralization is complex (Wdliams et al., 1960; Basak and Bhattacharya, 1962) and depends on the nature of the organic material (Fabry, 1963). In general, if aeration becomes poorer, the rate of decomposition of organic matter becomes smaller, an effect which becomes significant at oxygen levels below about 1% of the partial pressure of oxygen in the atmosphere (Greenwood, 1961). Thus Jackman (1964) observed that net immobilization of phosphorus occurred in the first 7.5 cm of the grassland soil and net mineralization of organic phosphorus occurred between 7.5 and 15 cm of soil depth. Apparently the effect was not due to the oxygen content of the soil but to the nature of the organic material in these layers. However, Campbell and Racz (1975), from their observations on greater organic P mineralization in flooded soils, suggested that anaerobic conditions increased mineralization rate. It would be interesting to investigate the effect of moisture and aeration on organic P mineralization separately and concurrently.

4. Soil p H The rate of mineralization is enhanced by adjusting the pH such that it is optimum for general microbial metabolism. Mineralization of soil organic phosphorus increases following liming of acid soils (Pearson et aZ., 1941;Goring and Bartholomew, 1952; Thompson et aZ., 1954; Halstead et al., 1963; Islam and Ahmed, 1973). The explanation for this phenomenon is that as the soil pH is increased, microbiological activity is markedly increased (Halstead et al., 1963), with concomitant increases in organic carbon and nitrogen mineralization. In addition, it may also be due to less organic matter being fixed by clays and the increase in the solubility of organic compounds. It has been observed that liming does not always increase mineralization of organic phosphorus (Kaila, 1961). Some of the variations in the effect of lime may be produced by the Ca:Mg ratio effect on mineralization and turnover of P in soil (Fabry, 1963). The rate of mineralization of organic phosphorus has been shown to be positively correlated with the mineralization rates of carbon and nitrogen. However, Thompson et al. (1954) showed that the rate of mineralization of organic phosphorus increased with increasing soil pH whereas the mineralization rates of carbon and nitrogen did not. A consequence of this is that in alkaline soils the ratios of organic carbon:organic phosphorus and total nitrogen:organic phosphorus should be greater than in acidic soils.

105

SOIL ORGANIC PHOSPHORUS

5. Addition of Inorganic Phosphorus It was indicated earlier that addition of inorganic phosphorus (e.g., superphosphate) to soils under pasture results in increasing immobilization of phosphorus (Jackman, 1964; Rixon, 1966). However, there are data (Table XIV) showing that the addition of inorganic phosphorus results in increased mineralization of organic phosphorus (McCall et d.,1956; Kaila, 1961; Acquaye, 1963; Fabry, 1963; Enwezor, 1966). The increase in the mineralization of organic phosphorus in the presence of inorganic phosphorus may be due to the increase in solubility of organic phosphorus; hence its susceptibility to mineralize because inorganic phosphorus competes with organic phosphorus for Fe, Al, and Ca that keep a part of organic P in a sparingly soluble form (Wier and Black, 1968). Some investigators, however, have observed that addition of inorganic phosphorus does not affect phosphorus mineralization. Thus Hofman and Teicher (1 964) found that additions of inorganic phosphorus as fertilizer had essentially no effect on the content of organic phosphorus in soil, Similarly, Wier and Black (1968) and Ghoshal (1975) could not find any evidence of enhanced mineralization of organic P due to the additions of inorganic phosphorus. It would be advantageous if the changes in different organic phosphorus fractions were studied following the addition of inorganic phosphorus rather than the changes in total organic phosphorus that may occur. It may also be helpful in estimating the labile organic phosphorus turnover in soil. Moreover, the concurrent changes in organic carbon, organic nitrogen, and organic sulfur, along with organic P upon the addition of inorganic phosphorus with and without the addition of nitrogen and sulfur that occur in soil organic matter TABLE XIV The Average Effect of Ca(H, PO,), *H, 0 on the Mineralization of Organic Phosphorus in Eight Organic Soils' Added phosphorus (ppm)

Organic P mineralized b (%)

12.5 25.0 50.0 100.0 200.0

1.3 1.9 11.2 76.9 83.5

aCalculated from McCall e t aL, Soil Science Society of America Proceedings 20, 81-83 (1956). %oils were incubated for 4 months at moisture equivalent and at 267°C. Organic P mineralized (%) = 100 (organic P without added P - organic P with added P)/organic P without added P.

106

R. C. DALAL

should provide a better understanding of the effect of the addition of inorganic phosphorus on the organic phosphorus mineralization in pasture as well as in cultivated soils. 6. Fertilizers Other Than Phosphorus

Since organic matter contains carbon, nitrogen, phosphorus, and sulfur in fairly definite proportions (Williams et al., 1960), the deficiency of carbon, nitrogen, or sulfur in soil should result in smaller amounts of organic matter buildup even if inorganic phosphorus in soil is in sufficient amounts. This proposition has not been widely tested in cultivated soils but in legume-grass pastures, where atmospheric nitrogen is fured by legumes and phosphorus is supplied in superphosphate, the buildup of organic matter, hence organic phosphorus, may be limited by sulfur supply (Section V, A). In the nonlegume cropping system, the application of nitrogen results in greater immobilization of phosphorus (Barrow, 1960). Conversely, when nitrogen fertilizer is not applied and plants derive nitrogen from the mineralization of organic nitrogen, organic phosphorus is also mineralized since mineralization of organic nitrogen and organic phosphorus are similar processes (Section V, B and Table XII). The similar effect of sulfur supply or withdrawal on phosphorus immobilization or mineralization could occur (Table XV). However, from the TABLE XV Effect of Varying Nitrogen, Sulfur, and Phosphorus Supply on Phosphorus Mineralization' Percentage of P mineralized (+) or immobilized (-)b

S Experiment no.

N

(mg/bottle)

P

7 days

21 days

1

5 15

1.5 1.5

3.0 3.0

-20

-31

-10 -1 0

2

15 15

1.5 0.5

3.0 3.0

-61 -43

-40 -27

3

15 15

1.5 1.5

3.0 0.5

+3 3

6 7

+ 7

+17

'Calculated from Barrow (1960). 'Incubated at 27°C for 7 and 21 days. In experiments 1,2, and 3, N, S, and P supply were varied by varying the proportion of glycine, cysteine, and sodium pglycerophosphate, respectively. In experiments 1 and 2, P was supplied as K, H PO,.

SOIL ORGANIC PHOSPHORUS

107

limited data available at present, it is not possible to postulate the mechanisms of the processes of immobilization and mineralization of phosphorus as influenced by the application of nitrogen and sulfur fertilizers. There is an urgent need to investigate this phenomenon in cultivated as well as in pasture soils. Z Cultivation

It has been observed that cultivated soils generally contain less organic phosphorus than virgin soils (Thompson et al., 1954). It has been suggested that cultivation increases the aeration of the soil which in turn stimulates microbial activity and subsequently greater decomposition of organic matter (Clements and Williams, 1964). But not all the components of organic matter are mineralized to the same extent. Thus Williams and Lipsett (1961) found that 50-60 years of wheat cultivation in New South Wales resulted in the loss of 30% of organic carbon compared with 17% of organic P. Further, not all the organic phosphorus fractions mineralize at the same rate, for Williams and Anderson (1968) observed that the decrease in organic phosphorus resulted from cultivation was mainly a decrease of inositol phosphates. The cultivation of a virgin thick chernozem resulted in the decrease of phytate, phospholipids, and nucleic acid-P compounds (Levenets and Krivonosova, 1974) and in increase in the organic phosphorus content in the nonhydrolyzable residues of the organic matter (Krivonosova, 1972; Batsula and Krivonosova, 1973). The land use also has an effect on the mineralization of organic phosphorus in soil, possibly by manipulating temperature, moisture, and aeration effects. Generally, the clean cultivated crops, such as maize, result in greater reduction in organic phosphorus compared with pastures. 8. Soil Microorganisms The rate of mineralization of organic phosphorus depends largely upon the population as well as the activity of microorganisms in soil. Microorganisms capable of extensively decomposing organic insoluble phosphorus compounds have been reported to be present in soil. Species of Aspergillus, Penicillium, Mucor, Rhizopus (Casida, 1959; Irving and Cosgrove, 1972) and BacilZus and Pseudomonas (Kobus, 1961;Cosgrove, 1970) produce phosphatases that degrade glycerophosphates, nucleic acids, and phytin. Up to 100% of the phosphorus in phytin and 50% in nucleic acids and certain phospholipids is ultimately dissolved by many bacterial strains (Kobus, 1961). Carbohydrate is apparently required as an energy source for degradation of organic phosphates in soil by microorganisms (Hannapel et a]., 1964b); hence, organic phosphorus can be mineralized rapidly near plant roots (Greaves and Webley, 1965). Moreover, phosphorus deficiency causes a 4- to 12-fold increase in phosphatase in many

108

R. C. DALAL

organisms, such as Escherichia coli (Torriani, 1960). Saccharomyces (Suomalainan et al., 1960), Euglena gracilis (Blum, 1965), and Neurospora crassa (Nyc, 1967). It should be emphasized that although the microorganisms are capable of decomposing organic phosphorus compounds the mineralization rates in soil are slow because soil organic phosphorus compounds are present in complex and largely insoluble forms (Hance and Anderson, 1963). Attempts have been made to exploit the microbiological release of phosphate to increase crop yields (Pikovskaia, 1948; Uarova, 1956; Cooper, 1959). Phosphobacterin (Bacillus megatherium var. phosphaticum) inoculum has been claimed to increase the yield of oats, wheat, millet, corn, and soybeans in the Soviet Union. However, Smith er al. (1961) could not confirm the results. Since many organisms similar to B. megatherium are already present in soil, the addition of these organisms can not be expected to have any appreciable effect. 9. Presence of Plants Hayashi and Takijima (1955), Sekhon and Black (1969), Thompson and Black (1970), and Somani and Saxena (1971) have all shown that the presence of plants decreased the organic phosphorus content of soil near their roots. Thompson and Black (1970) ascribed this effect to the transfer of substances in solution between soil colloids and roots (Table XVI). It is possible, as shown by Hannapel et al. (1964a), that mineralization of organic phosphorus could occur because of the addition of readily assimilable organic substances excreted by the roots (Barber and Martin, 1976). Ghoshal (1975) and Ghoshal and Jansson (1975) have shown that the addition of glucose stimulates the organic phosphorus mineralizing microflora. On the other hand, it is known that plant roots produce phosphatase enzymes (Greaves and Webley, 1965; Shaykh and Roberts, 19741, capable of dephosphorylating organic phosphorus. Moreover, phosphorus deficiency in soil could cause a considerable increase in phosphatase activity in the rhizosphere; Bieleski and Johnson (1972) observed that phosphatase activity of the roots of Spirodela oligorrhiza increased by 4-5 times under phosphorus deficiency conditions (Table VII). It has also been postulated that the greater uptake of phosphorus in P-deficient soils by plants in association with ectomycorrhiza (Harley, 1969) and endomycorrhiza (Endogone sp.) (Daft and Nicolson, 1969; ROSS,1971; Hayman and Mosse, 1971; Mosse et al., 1973; Khan, 1975) may be due at least in part to the greater phosphatase activity of the mycorrhizal plants (Table VIII); in addition to the increase in root surface area and increased volume of soil exploited by radiating hyphae (Harley, 1969). Since ectomycorrhiza and endomycorrhiza have similar physiological roles (Bieleski, 1973) it is suggested the Endogone-infected plants could utilize organic phosphorus especially from soils high in organic phosphorus, such as

109

SOIL ORGANIC PHOSPHORUS

TABLE XVI Organic Phosphorus of Soil Before and After Incubation for 5 Weeks in the Presence and Absence of Corn Plants' ~~

~~~

Organic P (pg/culture) Experiment

Original soil

With plants

Without ulants

1 2

25 1

220 25 0

237 26 2

283

'From Thompson and Black (1970). Soil was enclosed in Teflon-coated, glass-fiber filter paper.

pastures, similar to the action of beech mycorrhiza (Bartlett and Lewis, 1973) and endophyte mycorrhiza of CaZZuna uulgaris (Pearson and Read, 1975). However, this has not been confirmed so far (Hayman and Mosse, 1972).

C. MINERALIZATION OF ADDED ORGANIC MATTER

The mineralization of added plant residues has been well documented but the precise biochemical processes in the decomposition of added organic matter in soil have not been studied in detail because of the lack of suitable analytical techniques (Russell, 1973). In many experiments on the decomposition of plant residues in soil, it is mainly the carbon and nitrogen mineralization which are investigated; the mineralization of the phosphorus and sulfur constituents of the organic matter are rarely studied. A number of organic phosphorus compounds including nucleic acids, lecithin, and phytin are dephosphorylated when added to soil (Cosgrove, 1967). Pearson ef al.. (1941) studied the decomposition of lecithin (soybean meal), nucleic acid (RNA), and phytin (Table XVII). TABLE XVII Mineralization of Organophosphate in Soil' Acid-soluble inorganic P (ppm) Soil amendment

Organic P

Ob

Sb

4Sb

90b

Lecithin RNA Phytin

0.526

28.5 25.5 25.5

32.0 79.0 24.0

41.5 56.0

45.5 62.5 65.0

7.80 19.10

40.0

'Reproduced from Soil Society ofAmerica Proceedings, Volume 6, pages 168-175, 1941, by permission of the Soil Science Society of America. bIncubation days.

110

R. C. DALAL

Microbial populations of soil rapidly degrade added nucleotides (Cosgrove, 1967) and nucleic acids (Pearson et aL, 1941). Organic phosphorus in added organic matter, however, is gradually mineralized in soil. Birch (1961) found that no pronounced mineralization of plant organic phosphorus takes place during the first 3 months of decomposition. Fuller et al. (1956) studied the several factors that influence the rate of mineralization of phosphorus from crop residues added to soil. The phosphorus content of such plant materials has been found to be the important factor in determining whether net mineralization or immobilization of phosphorus occurs. The critical level above which mineralization takes place is about 0.2% (Section V, A). Kaila (1949), following culture studies, suggested that net mineralization would take place if the phosphorus content of the organic matter exceeded 0.3%. But in natural materials not all the organic carbon is readily available for utilization by microorganisms. Thus the quantity of phosphorus immobilized would be diminished, reducing the total carbon available for utilization, This could at least partly explain the conflicting results reported in the literature about the optimum C:P ratio in organic materials below which net mineralization of organic phosphorus would occur (Barrow, 1960; Hannapel et al,, 1964b; Enwezor, 1967). Moreover, phosphorus present in plant materials is not all organic. In fact, a high proportion of plant phosphorus may be inorganic (Birch, 1961;Bromfield and Jones, 1972); it is this inorganic fraction that supports the metabolic and synthetic processes of the microorganisms in the initial stages of decomposition. Birch (1961) has shown that no pronounced mineralization of plant organic phosphorus takes place during the first 3 months of decomposition (Section V, A). However, Martin and Cunningham (1973) observed that phosphorus could be released from dead plant roots before an extensive population of microorganisms has developed in response to the fresh organic matter. They suggested that the degradation of organic phosphorus in roots could result from the autolytic activity of plant enzymes. Later, Birch (1964) found that there are some microorganisms which are completely dependent on organic phosphorus as a source of phosphorus. Organic phosphorus is evidently only slowly mineralized but like inorganic phosphorus in added organic matter, .eventually becomes a part of the soil biomass as a constituent of microbial tissue and thus enters the cycle of phosphorus turnover in soil. It would be interesting to study these phosphorus constituents in the added organic materials and soil organic matter that are involved in the rapid turnover of organic phosphorus in soil. In addition to the carbon and phosphorus contents of the added organic matter, nitrogen and sulfur (Table XV) and perhaps other nutrient elements also determine the rate of mineralization of organic materials in soil; since it has been observed that a decreased supply of one element results in the increased mineralization of other elements (Barrow, 1960; Tisdale and Nelson, 1975).

111

SOIL ORGANIC PHOSPHORUS

Fuller et al. (1956) observed that in addition to phosphorus content, the maturity of the crop residues, the part of the plant added, the rate of application, and the length of time the organic materials have been added to soil also influenced the rate of mineralization of phosphorus from the added plant materials. Recently, Enwezor (1976) observed that the rate of phosphorus mineralization would be faster when a grass-legume mixture was added to soil than when either grass or legume alone was added. Soil conditions that favor the rapid decomposition of plant materials such as optimum N, proper aeration, moisture supply, and temperature (30°-45"C), also increase the rate of mineralization of phosphorus from added organic matter. The interdependent effects of these factors on added organic matter decomposition should be investigated (Wildung et al., 1975). The phosphorus applied to soil as animal manures represents a significant proportion of total applied P under certain conditions. Animals grazing on pastures return to the soil a major portion of the phosphorus they consume (Table XVIII). The greater part of the total fecal P is in inorganic form (Bromfield, 1961) which can be readily utilized by plants. Moreover, digestion by animals results in rapid as well as extensive mineralization of plant P (Table XIII). It is proposed that less labile plant organic P (possibly in phytates), is transformed into more labile microbial organic P in the rumen of animals [microbial P is largely nucleic acid-P (Barnard, 1969) and phospholipid-P (Bucholtz and Bergen, 1973)], although not enough evidence is available to confirm it. It has been shown, however, that a large fraction of the organic phosphorus of the animal manures is resistant to mineralization (Peperzak et al., 1959; Bromfield, 1961 ; Gunary, 1968), although eventually all organic phosphorus in manures is mineralized. Bromfield (1961) found a readily mineralizable organic fraction in sheep dung but the chemical nature of the mineralizable fraction or the resistant fraction is not known. TABLE XVIII Phosphorus Budget of an Established Pasture for a Year' System

Phosphorusb (kdha)

In soil (0-5 cm) Taken up by plants Consumed by animals Removed in product Returned to soil

170 26 25 2 23

%dculated from Till ef al. (1975). bAbout 23 kg P/ha (250 kg superphosphate) was applied to white clover (Trifoliurn repens)-carpet grass (Axonopus affinis) pasture and grazed at the stocking rate of 2.5 animals per hectare.

112

R. C. DALAL

It should be mentioned that although a major fraction of organic phosphorus in the manures is not readily mineralized, it plays an important role in the movement of phosphorus (probably in the microbial biomass) in the soil (Campbell and Ebcz, 1975). Since organic P moves more rapidly in soil than inorganic phosphorus (see Rolston el al., 1975, for earlier work), Rolston et al. (1975) proposed that organic phosphate compounds, such as glycerophosphate, glycolphosphate, methyl and ethyl ester phosphate, glucose-1-phosphate, and glucose6-phosphate, may prove to be more efficient fertilizers than inorganic orthophosphates (e.g., superphosphate). However, organic phosphates may contribute significantly to environmental pollution (see Griffith, 1973).

VI. Conclusions

Accumulation of organic phosphorus in soil is primarily a result of microbial activity. However, little is known of the processes involved in and the mechanisms of the incorporation of phosphorus in soil organic matter. Some investigations have been carried out on the accumulation of organic phosphorus under pasture and the incorporation of added inorganic phosphorus (superphosphate) in soil organic matter. It is generally agreed that organic phosphphorus is made available to plants largely after its mineralization into inorganic phosphorus although the water-soluble soil organic phosphorus (especially the hydrolyzable fraction) may be absorbed by plants directly, or after being dephosphorylated by the phosphatase enzymes near the root. The conflicting results reported on the availability of organic phosphorus to plants could be ascribed to the lack of knowledge about the chemical nature of added as well as soil organic phosphorus. About half of the soil organic phosphorus has been characterized so far. Inositol phosphates certainly dominate, while nucleic acids and their derivatives and phospholipids comprise only a small fraction of soil organic phosphorus. Studies on the immobilization and mineralization of phosphorus are further complicated by the fact that the phosphorus fraction in the organic matter varies much more than the nitrogen and sulfur fractions. Therefore, the organic carbon:organic phosphorus ratio, unlike organic carbon:total nitrogen and organic sulfur ratio, is an unreliable index of phosphorus mineralization. The cause of the greater variation in the organic carbon to organic phosphorus ratio awaits further studies. In spite of the variability in the C, N, S, and P constituents of the organic matter, the mineralization as well as immobilization processes of N, S, and P are essentially similar although not enough is known about the processes. It is important to study the processes that govern the turnover of carbon, nitrogen, phosphorus, and sulfur in soil organic matter, for organic matter supplies these nutrient elements in significant amounts to the pastures and arable crops, especially in warmer climates.

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113

ACKNOWLEDGMENTS

The author is grateful to Dr. G. J. Blair for permission to use his unpublished data, and to Dr. G. Anderson and Professor A. Lazenby for their helpful suggestions. Financial assistance was provided by the Australian Meat Research Committee. REFERENCES Acquaye, D. K. 1963. Plant Soil 19,65-80. Adams, A. P., Bartholomew, W. V., and Clark, F. E. 1954. Soil Sci. Soc. Am., Proc. 18, 4W6. Alexander, M. 1961. “Introduction to Microbiology,” pp. 353-369. Wiley, New York. Anderson, G. 1960. J. Sci Food Agric, 9,497-503. Anderson, G. 1961. J. Soil Sci 12,276-285. Anderson, G. 1964. Trans. Inr. Congr. Soil Sci., 8th. Bucharest pp. 563-512. Anderson, G. 1967. In “Soil Biochemistry” (A. D. McLaren and G. H. Peterson, eds.), pp. 67-90. Dekker, New York. Anderson, G. 1970. J. Soil Sci. 21,96-104. Anderson, G., and Arlidge, E. 2,1962. J. Soil Sci. 13, 216-224. Anderson, G., and Hance, R. J. 1963. Plant Soil 19, 296-303. Anderson, G., and Malcolm, R. E. 1974. J. Soil Sci. 25, 282-297. Anderson, G., Williams, E. G., and Moir, J. 0. 1974. J. Soil Sci. 25, 5 1-62. Baker, R. T. 1976. J. SoilSci. 21, 504-512. Barber, D. A., and Martin, J. K. 1976. New Phyfol. 76,69-80. Barnard, E. A. 1969. Nature (London) 221,340-344. Barrow, N. J. 1960. Ausf. J. Agric. Res. 11, 317-330. Barrow, N. J. 1961. Soils Ferf. 24, 169-173. Bartlett, E. M., and Lewis, D. H. 1973. Soil Biol. Biochem. 5,249-257. Basak, M. N., and Bhattacharya, R. 1962. Soil Sci. 94, 258-262. Batsula, A. A., and Krivonosova, G. M. 1973. Pochvovedenie No. 6, pp. 24-27. Bieleski, R. L. 1973. Annu. Rev. Plant Physiol. 24, 255-252. Bieleski, R. L., and Johnson, P. N. 1972. Aust. J. Biol. Sci. 25, 707-720. Birch, H. F. 1961. Plant Soil 15,347-366. Birch, H. F. 1964. Planf Soil 21, 391-394. Birch, H. F., and Friend, M. T. 1961. Nature (London) 191, 731. Black, C. A. 1968. “Soil-Plant Relationships,” pp. 558-653. Wiley, New York. Black, C. A., & Goring, C. A. I. 1953. In “Soil and Fertilizer Phosphorus in Crop Nutrition” (W. H. Pierre and A. G. Norman, eds.), Agronomy, Vol. 4, pp. 123-152. Academic Press, New York. Blum, J. J. 1965. J. Cell Biol. 24, 223-234. Bowen, G. D. 1973. In “Ectomycorrhizae. Their Ecology and Physiology” (G. C. Marks and T. T. Kozlowski, eds.), pp. 151-205. Academic Press, New York. Bromfield, S. M. 1961. Aust. J. Agric. Res. 12, 111-123. Bromfield, S. M., and Jones, 0. L. 1972. Ausf. J. Agric. Res. 23,811-824. Bucholtz, H. F., and Bergen, W. G. 1973. Appl. Microbiol. 25,504-513. Caldwell, A. G., and Black, C. A. 1958. SoilSci. Am. Proc. 22, 296-298. Campbell, L. B., and Racz, G. J. 1975. Can. J. SoilSci. 55,457-466. Carter, E. D. 1958. Proc. Aust. Agrosf, Conf: 1, 33-35. Casida, L. E., Jr. 1959. SoilSci. 87, 305-310.

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GROWTH OF THE LEGUME SEEDLING' C. S. Cooper Agricultural Research Service, United States Department of Agriculture, Bozernan, Montana

I. Introduction .................................................. 11. Physiological Predetermination .................................... 111. Germination ................................................... A. Hard Seed .................................................. B. Temperature ................................................ IV. Stages of Seedling Development .................................... A. Heterotrophic Stage .......................................... B. Transitional Stage ............................................ C. AutotrophicStage ............................................ V. Improvement of Legume Seedling Vigor ............................. VI. Seedbed Preparation ............................................ VII. Seeding Forage Legumes ......................................... A. Seed Treatment ........................ ..................... B. Calculation of Seeding Rates ................................... C. DrillCalibration ............................................. D. Seeding .................................................... E, Sodseeding ................................................ VJII. Seeding Management Practices ..................................... References . . . . . . . . . ...... .............................

119 120 121 121 122 123 123 125 1 26 130 130 131 131 131 133 134 134 137 137

I. Introduction

Successful stand establishment is of major importance for the profitable use of legumes in hay and pasture plantings. Because of their small seed size, forage legumes are more difficult to establish than are larger seeded legumes such as peas (Pisurn spp.) or beans (Phaseoh spp.). Failure to establish stands is not uncommon despite the accumulation of knowledge concerning seedling growth and the development of sophisticated seeding equipment as an aid to seeding success.

' Contribution from the Western Region Agricultural Research Service, USDA and the Montana Agricultural Experiment Station. Published with approval of the director as paper no. 696. 119

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C. S. COOPER

The growth of a legume seedling depends on its inherent vigor and the environmental conditions present during seed germination, maturation, and growth. During this century there has been much research concerning growth of the legume seedling. With the exception of review papers dealing with the effect of seed size upon seedling growth, however, few attempts have been made to summarize these data. This paper is concerned with factors affecting the development and growth of the forage legume seedling. I I. Physiological Predetermination

Fifty-six years ago Kidd and West (1919) pointed out that environmental conditions during seed formation could influence seed size and subsequent progeny performance. They designated growth responses which could be traced to environmental conditions at some stage of previous development as “physiological predetermination” in order t o distinguish them from those which are due to hereditary causes. They listed three conditions which could affect the “potentiality” of the seed or the capacity of the resulting plant for growth and yield. These were: (1) parental conditions; (2) conditions immediately preceding germination, during germination, or in the early stages of the seedling’s growth; and (3) harvesting conditions. The most noticeable manifestation of physiological predetermination is seed size. Seed size may be affected by the position of seed on the plant, the amount of substrate and nutrients available during seed formation, and the environment during seed development. Seed size varied from 2 to 34 mg in one strain of subterranean clover (Black, 1957) and from 2.7 to 24.6 mg in one strain of sainfoin (Fransen and Cooper, 1976). Similar ranges of seed size are observed in most forage legume species. Legumes such as birdsfoot trefoil or crown vetch with indeterminate flowering habit experience a greater range of environmental effects during seed development than those with a determinate flowering habit. Anderson (1955) reported that umbels of birdsfoot trefoil set in early season produced more pods and usually more seeds per pod than umbels set in late season. Time of harvest may also affect seed size and embryo maturity. With determinate species, harvest may be timed to occur when most seeds are mature, but indeterminate species display seeds of various stages of maturity at any given harvest date. Immature seeds are usually smaller and of lower viability than mature seeds (Anderson, 1955; Carleton et al., 1967). Birdsfoot trefoil seed reaches maximum dry weight at 35 to 40% moisture (Anderson, 1955) and sainfoin seed at 40% moisture (Carleton et al., 1967). Maximum dry weight for most forage legume seeds probably occurs at 35-40% moisture. Early seedling growth is proportional to seed size in alfalfa (Beveridge and Wilsie, 1959; Carleton and Cooper, 1972), birdsfoot trefoil (Carleton and

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Cooper, 1972; Hensen and Tayman, 1961; Stitt, 1944; Twamley, 1967), sainfoin (Carleton and Cooper, 1972; Cooper, 1966), and subterranean clover (Black, 1956, 1957, 1959). Fransen and Cooper (1976) showed that seedlings from large sanfoin seeds emerged and developed first and second leaves faster than seedlings from small seeds. Embryo axis and leaf primordia length were directly proportional to seed size. The more rapid seedling growth from larger seeds during the heterotrophic phase is probably due to a more advanced stage of embryology rather than to more stored reserves. Environmental conditions during maturation may affect the performance of seed. Dotzenko et al. (1967) found that alfalfa seeds produced under a wide range of temperatures in the field displayed significant variation in the percentage of hard seed, quick germination, and total germination. In general, hard seed content is greatest when seed is produced in cooler climates (Gunn, 1972) and is greater in small seeds within a species than in larger seeds (Black, 1959). Although climate during maturation may affect hard seed content and size of seed, it does not necessarily affect the subsequent growth of the seedling. Cooper (unpublished data) found no difference in relative growth rate, net assimilation rate, or leaf area ratio of seedlings grown from alfalfa seed produced from an identical 2-clone cross in Arizona, Montana, and Nevada. In his work, seeds were screened to remove seed size effects which may have occurred as a result of environment.

I I I . Germination

A. HARDSEED

Legumes imbibe water much more rapidly than grasses and nearly all of the water needed for germination is imbibed during the first 4-8 hours (McWilliam e t aZ., 1970). The presence of a seed pericarp slows the rate of imbibition in crimson clover (Stitt, 1944) and in sainfoin (Carleton et al., 1968). Hard seeds are impermeable to water and thus incapable of imbibition until the seed coat is scarified. Many legumes contain a large percentage of hard seeds if hand-harvested, but are scarified to some degree in the process of threshing. Rincker (1954) reported an average of 64% hard seed for 66 samples of Wyoming machine-threshed certified alfalfa seed, and Cooper (1957) reported a hard seed content of 80 t o 90% for an annual native clover in Oregon. Hard-seed content of crown vetch (Brant et ab, 1971) and of cicer milk vetch (Carleton et al., 1971) may be more than 75%. Impermeability in legume seed is ascribed to the cuticle or the macrosclerid layer, also known as the pahade layer or the Malpighian layer (Brant et aZ., 1971). Rincker (1954) made hard seeds of alfalfa permeable by exposing them to a temperature of 105°C for 90 seconds or to 42°C for 1 hour. Barton (1947)

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made seeds of whlte sweet clover (Melilotus alba Desr.) absorb water by plunging them into liquid N (-1953°C). Brant'et al. (1971) successfully scarified seed of crown vetch by: (1) immersion in 18 N sulfuric acid for 15 minutes, (2) immersion in boiling hot water for 15 seconds, followed by a dip in cold water, (3) mechanical scarification, and (4) two 2-minute immersions in liquid N. They also reduced hard-seed content slightly by treatment with hemicellulase and pectinase. The degree of scarification required differs with species and among seed lots within a species. Carleton et al. (1971) reported that cicer milk vetch required much more intense mechanical scarification than alfalfa. They developed a "quick swell test" for determining the effectiveness of scarification. Following scarification treatment, seeds are germinated on wet blotter paper. After 24 hours, the percentage of seeds swollen is determined. They reported that 30 to 50% swollen seeds is commensurate with good scarification. Higher percentages were often associated with a high degree of seed injury which resulted in poor emergence when seeds were planted.

B. TEMPERATURE

Most forage legume seeds germinate over a wide range of temperatures but the optimum germination temperature varies among species. Those species which are easiest to establish in the field also have the ability t o germinate completely and rapidly over a range of temperature (Townsend and McGinnies, 1972). Townsend and McGinnies (1972) grew a number of legume species at three alternating temperatures (5"-2OoC, 1So-25"C, and 2Oo-25"C) and at a constant 2OoC. Duration of alternating temperatures was 12 hours. They considered alfalfa and sainfoin to be temperature insensitive for total germination over the range of temperatures tested. Germination rate and total germination of cicer milk vetch increased through 15"-25"C but decreased at higher temperatures. McElgunn (1973) germinated sweet clover, alfalfa, birdsfoot trefoil, and sainfoin at constant temperatures of 7", lo", 13", or 21°C and 12-hour alternating temperatures of 2"-13OC, 4"-15"C, 7"-18"C, or 16"-27OC. Rate of germination averaged across all temperatures was in the order of sweet clover > alfalfa > birdsfoot trefoil > sainfoin. He concluded that cold alternating temperatures reduced both germination rate and total germination. Sainfoin had the slowest germination rate when averaged across all temperatures. For the first 5 days, germination rate at constant temperatures was faster than at alternating temperatures for all species. Qualls and Cooper (1968) found that respiration and germination rate of birdsfoot trefoil increased with increasing temperatures from 15.6" t o 26.7"C. They found differences in germination rate of seeds of the same size from different varities. Carleton et al. (1968) showed that sainfoin

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seed germinated best at a temperature range from 15" to 23°C but germination decreased at 30" or 35°C. During the first 8 days of germination, seedling length increased most at 20" to 30°C and least at 35°C. Young et al. (1970) found that subterranean clover, woolypod vetch (Vicia dasycarpa Ten. var. Lana), cup clover (Trifolium cherileri L.), and sainfoin had a remarkable amount of germination at 5" and 0.5"C. They state that these legumes appear to be hghly adaptable t o germination in late fall or early spring in cold seedbeds of arid rangelands. McWilliam et al. (1970) found that germination rate of whte clover and alfalfa increased from 5" to 30°C but germination of subclover declined markedly at temperatures above 30°C.

IV. Stages of Seedling Development

Once a seed germinates, the resultant seedling goes through three stages of development which have been defined by Derwyn et al. (1966) as (1) heterotrophic, (2) transitional, and (3) autotrophic. For a legume seedling, the heterotrophic phase is from imbibition of water until emergence and commencement of photosynthesis in cotyledons. The transition stage occurs when cotyledons begin to photosynthesize but before the exhaustion of reserves. The autotrophic stage follows the exhaustion of the cotyledonary reserves. At this time the seedling is entirely dependent upon photosynthesis and is a true autotroph.

A. HETEROTROPHIC STAGE

During the heterotrophic stage of seedling development, the rate of transfer of stored reserves to the embryo axis is highly dependent upon temperature (Derwyn et al., 1966). Rapid extrusion of the seedling root is important to establishment, particularly where surface soils dry rapidly, or where conditions favorable to germination may be of short duration such as on arid rangelands. Rate of root extrusion is more rapid for annuals than for perennials and is a factor in the capacity for regeneration, which permits the use of annuals in pastures (McWilliam et al., 1970). Rate of seedling elongation varies within a species and is directly correlated with seedling vigor (Qualls and Cooper, 1968). The rate of hypocotyl elongation often determines the duration of the heterotrophic stage of development because cotyledons begin photosynthesis upon emergence and the seedling enters the transition stage. Size of seed and depth of seeding are two major determinants of emergence rate. Fransen and Cooper (1976) studied growth and development of sainfoin seedlings from four seed sizes of 10 sainfoin accessions. In all accessions, seedlings from large seed emerged earlier and developed more rapidly than seedlings from small seed.

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Black (1956) reported emergence of subterranean clover seedlings from medium and large seed 1 day earlier than seedlings from small seed when sown at a depth of 3.2 cm. Jensen et al. (1972) reported that the emergence force of seedlings from large-seeded alfalfa, red clover, alsike clover, and narrow leaf birdsfoot trefoil was greater than that for small seeds. Williams (1956) found that differences in emergence force of seeds from several legume species was directly related to seed size. Depth of seeding is of major importance to emergence and establishment. Stickler and Wassom (1963) obtained 53, 25, and 18% emergence of birdsfoot trefoil from planting depths of 1.0, 2.5, and 3.8 cm, respectively. Moore (1943) found best emergence of red, crimson, and alsike clover, white and yellow sweet clover, alfalfa, and several Lespedeza spp. from a depth of 0.6 or 1.3 cm. Seedlings from deeper plantings emerged more slowly and were less vigorous. Erickson (1946) found that a 0.6-cm depth was most favorable for small alfalfa seeds and that a 1.9-cm depth was best for large alfalfa seeds. He suggested a 1.3-cm depth as a desirable compromise. Peiffer et al. (1972) found that ‘Penngrift’ crown vetch had similar emergence from soil depths of 1.3, 1.9, and 2.5 cm but reduced emergence at 3.8 cm. Birdsfoot trefoil emergence decreased at 2.5 cm and red clover and alfalfa at 3.8 cm. For most forage legumes, seeding depth should be no greater than 1.3 cm. Deeper planting may appear to be advantageous in order to place seeds in a moist soil. However, deeper planting often results in weakened seedlings and prolongs the period when seedlings are most susceptible to disease. Depth of seeding may be important in terms of seedling competition. In mixtures, some seeds will have an advantage over others at a given seed depth. Stapledon and Wheeler (1948) concluded that optimum establishment of herbage seeds could follow only from sowing the different fractions of a seed mixture at depths suited to the individual seed size. Such a practice would be difficult with most commercial seeding equipment. During the heterotrophic stage both the amount and rate of germination may be affected by osmotic concentration. Uhvits (1946) germinated alfalfa seeds in substrates supplied with NaCl and mannitol at osmotic pressures ranging from 1 to 15 atmospheres. The rate and percentage of germination decreased as the osmotic concentration increased. Germination was practically inhibited at 12 to 15 atmospheres of NaCl. Up to 9 atmospheres, however, alfalfa germination was 83% after 10 days of germination compared to 88% for tap water. Similar results were obtained with a number of legumes germinated at a range of osmotic concentrations by Young et al. (1970). The legume seedling can assimilate and use externally supplied nutrients at an early age. McWilliam e f al. (1970) reported an increase in weight of legumes receiving nutrient solutions 5 days after imbibition. They reported six- to tenfold increases in nitrogen content during the first 12 days of

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growth. Subterranean clover absorbed 32P as early as 4 days after imbibition began.

B. TRANSITIONAL STAGE

When the cotyledon emerges, the seedling derives energy from photosynthesis as well as from stored reserves. The duration of the transitional stage of forage legume seedling growth may be very short and is dependent upon the amount of reserves left at emergence. The quantity of reserves remaining at emergence is primanly affected by depth of planting (Black, 1955). Temperature affects rate of reserve utilization, which in turn determines rate of growth (QuaUs and Cooper, 1968). The role of the cotyledon as a storage organ ends with the complete utilization of reserves. At t h s time, the major role of the cotyledon is photosynthesis (Cooper and Fransen, 1974). McWilliam et al. (1970) detected photosynthesis in subterranean clover on the third day after imbibition and on the fourth day the compensation point between respired C 0 2 and accumulated COz was reached. Black (1955) found that the percentage of cotyledonary reserves remaining at emergence was dependent upon depth of seeding and temperature. When seeded at a 1.3-cm depth at a temperature of 2I0C, 61%of the reserves were present at emergence, but when seeded at a 5-cm depth and a temperature of 28OC, only 33% were present at emergence. He states that cotyledon reserves at emergence are not likely to limit seedling growth. In sainfoin, however, cotyledonary reserves decreased at the same rate when seeds were germinated in the dark or the light even though seedlings grown in light had green cotyledons by the third day (Cooper and Fransen, 1974). In this species, it appears that cotyledonary reserves are nearly completely utilized before photosynthesis begins. Stored energy in cotyledons of seedlings grown in darkness was not sufficient to allow normal first leaf formation. In some crops, such as corn, new leaf area formed must be able to supply energy at a rate equivalent to the energy derived from endosperm if seedling growth is t o progress normally (Cooper and MacDonald, 1970). Removal of part of the endosperm will decrease growth. During the transfer of cotyledonary reserves t o the seedling axis, total weight of the seedling decreases due to weight losses from respiration until reserves are utilized. Sainfoin lost 38% of its weight from imbibition until 9 days of age at a temperature of 20°C (Cooper and Fransen, 1974). Greenhouse-germinated birdsfoot trefoil seedlings lost more weight when grown under low light intensity than under high (Lin, 1963). The amount of dry matter lost varied from 25 to 45% among different varieties grown under 25% of greenhouse light intensity. The efficiency of conversion of stored substrate to new growth, calculated by dividing new growth by weight of dry matter lost from cotyledons of endo-

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sperm, varies among crops. Conversion efficiencies of 56% (Cooper and Fransen, 1974), 65% (Cooper and MacDonald, 1970), and 57% (Cooper, unpublished data) have been reported for sainfoin, corn, and barley, respectively, when grown in the dark. Cotyledons have not been extensively studied in forage legumes. In other species, epigeal cotyledons have been shown to vary in size and shape and in the length of time in which they become functional (Love11 and Moore, 1971). Some plants such as cucumber (Cucumis sativus L.) develop leaflike structures with high photosynthetic rate. Others such as Phaseolus spp. have thick cotyledons which senesce rapidly following utilization of reserves. Forage legume cotyledons are between these two extremes, but they differ in size and thickness and probably in rate of senescence among species. In alfalfa, birdsfoot trefoil, and sainfoin, each milligram of seed weight produced a cotyledonary area of 14.0, 10.1, and 3.5 mm, respectively (Lin, 1963). In studies with birdsfoot trefoil (Lin, 1963), subterranean clover (Black, 1957), and sainfoin (Fransen and Cooper, 1976), however, the ratio of cotyledonary area to seed size within a species appears nearly constant. Within a species, seed size is the major factor affecting the size of the cotyledon. The initial growth of legumes in a newly seeded field is dependent upon the amount of cotyledonary area present per unit of land surface. The quantity of cotyledonary area in turn is related to the number of seedlings per unit of land area and to the size of seed. The contribution of the cotyledon in total seedling photosynthesis decreases as total leaf area increases (Cooper and Fransen, 1974). Differences in cotyledonary senescence which might occur among different forage species have not been studied. Opik and Simon (1963, 1966) state that cotyledon senescence begins as food reserves are utilized. At this time, cotyledons have a maximum water content, but dry weight and respiration rate have declined rapidly. Chloroplasts begin breaking down, leaving the nucleus with an irregular membrane. Cotyledons turn different hues of yellow owing to breakdown of chlorophyll, allowing the carotenoids to become visible. Finally, an abscission layer forms between cotyledons and the remaining plant, and cotyledons are shed.

C. AUTOTROPHIC STAGE

Upon exhaustion of food reserves within the cotyledon, the legume seedling becomes a true autotroph. Its ability to establish itself and to compete with weeds and other crop species is dependent upon inherent seedling vigor and the effects of environment. Some species such as cicer milk vetch, crown vetch, birdsfoot trefoil, and

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sericia lespedeza, are slow to become established and often have thin unproductive stands because of competition from companion crops or weeds (Hensen and Tayman, 1961). Numerous studies have been reported relative to legume seedling response to light. Cooper (1966, 1967) using growth analysis techniques, studied growth responses of alfalfa and birdsfoot trefoil when grown under low light intensity in growth chambers and when grown under various levels of shading in the field. Under light intensities of 21 and 86 Klux in growth chambers, or 8 t o 100% of full sunlight in the field, birdsfoot trefoil had a higher relative growth rate than alfalfa. Thus, the poorer seedling vigor of birdsfoot trefoil was not due to differences in the effect of decreased light intensity on relative growth rate. He states that “although shading tolerance of birdsfoot trefoil and alfalfa may be similar, birdsfoot trefoil is more susceptible to being shaded, both in the seedling stage and later. Seedlings and mature plants of birdsfoot trefoil begin growth later in the spring and recover more slowly after clipping. These factors increase the likelihood of birdsfoot trefoil becoming shaded by associated species.” Gist and Mott (1958) reported that growth response of birdsfoot trefoil to low light was similar to alfalfa and red clover, but seedling growth was always less. Cooper (1966) found that the ability of alfalfa to outyield birdsfoot trefoil in the seedling stage is due entirely to initial seed size or, in terms of Watson’s (1952) definition of growth dependency, to “initial capital.” Shading or low light intensity affects distribution of dry matter into tops and roots. Cooper (1966) reported that less dry matter is partitioned into seedling roots with decreasing light intensity. As a result, shaded seedlings may become more susceptible to drought because of restricted root development (Cooper, 1966; Shirley, 1945). Cooper and Ferguson (1964) reported that rooting depth of birdsfoot trefoil was only 20 cm and alfalfa only 40 cm when the barley companion crop with which they were grown was harvested. At the same time, roots of both species had penetrated to a depth greater than 61 cm when grown without a companion crop. McKee (1962) found that birdsfoot trefoil shaded for 5 weeks required 6 to 11 weeks of growth in full daylight t o restore its original top/root ratio. He also found that the leaf area per plant of “Pennscott” red clover often increased with shading. Pritchett and Nelson (1951) reported that one of the most striking effects of reduced light intensity on alfalfa was the proportional decrease in nodulation. They found that nodulation ceases at less than 2.6 Klux and felt that this may contribute to loss of seedlings in the field. McKee (1962) reported that “Vernal” alfalfa and “Empire” and “Viking” birdsfoot trefoil required at least 25% of daylight to be functionally nodulated and 50% to be adequately nodulated. In contrast, functional nodules of Pennscott red clover were observed at 12.5% of fill daylight. Temperature has a marked effect on metabolic processes and consequently

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affects the growth of the legume seedling. Richards et al. (1952) state "Such physiological phenomena as cytoplasmic streaming, bio-electrical potential, synthesis of organic materials, translocation and respiration are all influenced by temperature." Temperature affects nutrient absorption. Potassium (K), nitrate, and bromide ion uptake increases with temperature increases from 6" to 30°C (Hoagland and Broyer, 1936). Temperature coefficients are higher for anion absorption than for cation absorption (Wanner, 1948). In alfalfa, K increased in tops and decreased in roots with increasing temperature, but Mg and Ca content of both tops and roots decreased with increasing temperature. Potassium content of soybeans increased with increasing temperatures of 12', 22", and 32"C, while divalent cation content decreased (Wallace, 1957). Each legume species has an optimum growth temperature (Stapledon and Wheeler, 1948). When temperatures exceed this optimum, roothop ratios are reduced, and when temperatures are less than optimum, root/top ratios are increased (Sprague, 1943). Smoliak et al. (1972) germinated and grew alfalfa, cicer milk vetch, and sainfoin with soil temperatures controlled at 7", 13", and 27°C for a 28-day period. Cicer milk vetch failed to emerge at 7°C and emerged and grew slowly at 18°C. Both alfalfa and sainfoin emerged and grew at 7°C. Alfalfa and cicer milk vetch developed best at 27°C but sainfoin grew equally well at 18" or 27°C. Late seeding at warmer temperatures seems to favor cicer milk vetch establishment, and sainfoin can evidently be established early in the spring with cool temperatures. McKell et al. (1962) studied growth response and phosphorus (P) utilization of four native and four introduced legumes at 10.Oo, 15.5", and 21.1"C. Root and top growth and P content increased with increasing temperature. Trifolium subterraneum and T. incamaturn grew better at 15.6"C than did the other six species. Four species, T. tridentatum, T. subterraneum, T. incamaturn, and Medicago hispida responded to P at 15.6" and 21.1"C better than T. variegatum, T. hirtum, T. microcephalum, and Lotus purshianus. Stimulation of root growth with P fertilization during cool winter months was suggested as a major factor in the subsequent production of these legumes. Many winter annuals of the genus Wfolium germinate rapidly at temperatures of 20°C or below but have limited germination above 30" to 35°C. The temperature environment of seedling legumes is largely dependent upon the climate of the area in which they are seeded. Temperature during the early stages of growth may be controlled to some degree by time of seeding, although extremes in temperature variation from year to year are likely to be the rule rather than the exception. The presence or absence of competition will also affect temperature of the microenvironment. Cooper and Ferguson (1964)

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found soil temperatures 1.6” to 4.4”C lower under a barley companion crop at soil depths of 7.6 to 61 cm. The presence of an overstory species such as a companion crop or weeds reduces the insolation intensity and influences the wavelength of radiation reaching the soil surface. Geiger (1950) reported that the insolation intensity of the soil surface under plants 1 meter high was only one-fifth that at the surface of bare ground. Reflectivity was 8-20% for visible light but rose to 45% for infrared (Geiger, 1950). Thus, both intensity and quality of light reaching the soil surface differ under plant cover and may influence soil and air temperatures near the soil surface. Geiger (1950) points out that the intensity of incoming radiation upon a growing crop and bare soil is the same during the day. Likewise, the intensity of outgoing radiation at night is equal. The effect of the plant cover is on the distribution of heat gained or lost, Dense stands of plants result in cooler air temperatures near the ground during the day. At night, however, outgoing radiation is from the top surface of the vegetation and the air is consistently warmer near the ground. In contrast, in the absence of an overstory species, radiation is from the soil surface, resulting in a cooler layer of air near the ground. As a result, legumes seeded with a companion crop have lower growth temperatures during the day but warmer growth temperatures during the night than legumes seeded in pure stands. Competition for the young legume seedling is generally from weeds or companion crops. Legume species differ in their abilities to compete, primarily as a result of differences in growth rate. These differences may affect the vegetative composition of those mixtures which contain more than one legume, since those legumes which compete best may have a greater survival rate under the stress of competition. Blaser er al. (1956) ranked aggressiveness of some common forage legumes as follows: Very aggressive Alfalfa (“Kansas common”) Red clover (“Kenland” or “Northern Neck”) Sweet clover Crimson clover Aggressive Alsike clover White clover (Ladino) Birdsfoot trefoil (“Granger” or “Italian”) Nonaggressive White clover (S-100, Virginia, and Lousiana) Birdsfoot trefoil (“Empire”) Big trefoil From that ranking, it is evident that the degree of aggressivenessvaries within a species as well as among species.

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Improvement of Legume Seedling Vigor

Seedling vigor of legumes is proportional to seed size. However, cultivars of a species with the same seed size may differ in vigor. Thus, in breeding for seedling vigor, both seed size and inherent seedling vigor must be considered. Birdsfoot trefoil has received much attention in breeding programs for seedling vigor, mostly because of difficulties encountered in establishing this species. Draper and Wilsie (1965) in three cycles of recurrent selection increased seed size of “Viking” birdsfoot trefoil 30% per cycle and of “Empire” 6% per cycle. Twamley (1974) increased seedling vigor of “Leo” birdsfoot trefoil 35 to 40% in three cycles of selection. In his program, selection was made for large-seeded plants and then for the most vigorous seedlings from seed of those plants. VI. Seedbed Preparation

The ideal seedbed for forage legumes should be weed-free and firm enough that a man walking across it does not leave a footprint deeper than 0.3 cm, and should have enough loose surface soil to cover seed to a depth of 0.6 to 1.2 cm deep (Cooper et al., 1973). Any tillage equipment that will provide this type of seedbed will be satisfactory. The moldboard plow turns under crop residue and makes the soil easier to prepare for seeding. It prepares a loose seedbed 10-25 cm deep, which requires additional treatment before it is ready for seeding. The major advantage of the moldboard is that it destroys existing vegetation and buries seeds of grassy weeds such as cheatgrass (Bromus tectorum L.) and foxtail (Hordeumjubacum L.) deep enough to prevent their germination and reestablishment. Disadvantages are the loose seedbed and a relatively high cost per hectare. Disk plows are not as effective as the moldboard in eliminating existing vegetation but are more effective under drier conditions in handling residue and shrubby growth. The one-way disk plow effectively covers heavy residue and controls weeds but leaves the soil loose. An offset disk is more effective than the one-way disk in breaking down sod and large clods and in killing small weeds. Following disking, harrowing will smooth the seedbed and firm the soil, although not to the degree needed for seeding. Spike harrows smooth the soil but leave it subject to wind erosion. Spring tooth harrows leave the seedbed less subject to blowing because they leave more clods on the surface. Most seedbeds require rolling or cultipacking to acquire the degree of firmness needed for the seeding operation. In the arid regions of the West, cultipacking is essential for obtaining a good firm seedbed. In the more humid areas of the Midwest and East, cultipacking during the spring is less necessary and may lead

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13 1

to excessive soil compaction (Tesar and Jackobs, 1972). Firming is best accomplished by rolling with a heavy roller or by cultipacking. If the needed equipment is not available for this, a weighted spike harrow or an irrigation float or leveler will compact the soil before seeding. VII. Seeding Forage Legumes

A. SEED TREATMENT

Before seeding, legume seed should be inoculated with symbiotic bacteria (Rhizobium spp .). The symbiotic bacteria are specific for many legumes such as birdsfoot trefoil and sainfoin but in some cases bacteria will cross-inoculate with several species. Inoculation is essential when a legume is seeded in an area for the first time, For successful legume inoculation the following procedures should be adhered to: 1. Select the proper inoculant for the legume t o be grown. 2. Store the commercial culture in a cool, dark place until it will be used. 3. Plant seed within 48 hours after inoculation, or reinoculate. 4. Inoculate in all cases of doubt, and always inoculate on new land. Small amounts of seed and inoculant may be mixed in a tub or bucket. Larger amounts may be mixed in a small concrete mixer or by hand on a cement floor or on the bottom of a truck bed. Addition of sticking agents such as milk or diluted syrup will help inoculant adhere to seed, Most companies that sell inoculant also sell sticking agents.

B. CALCULATION OF SEEDING RATES

Seeding rates are recommended to provide a given number of viable seeds per linear meter of drill row. The percentage of viable seed in a seed lot is calculated by multiplying germination percentage by purity percentage and dividing by 100. The value obtained is called pure live seed index (PLS). Thus if a seed lot has a percentage germination and purity of 90 and 85, respectively, the PLS is 90 X SS/lOO = 76.5%. For legumes, the percentage of hard seed is added to the germination percentage before multiplying it by the purity percentage. The number of seeds planted per meter of row is dependent upon the number of seeds per kilogram, the kilograms of seed planted per hectare (ha) and row spacing. It may be computed as follows: seed per meter of

= Number of PLS/kg X planting rate in Kg PLS/ha

row m in ha at width to be planted

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Thus if a species contains 220,000 PLS/kg, and a planting rate of 6 kg PLS/ha is desired with 15-cm row spacing, viable seed per linear meter of row would be: 220’ooo 66,666

=

19.8 viable seeds per row meter

The number of seeds that would be seeded per meter of row with a seeding rate of 1 kg/ha and with several row spacings is shown for some common forage legumes in Table I. These values are based on seeds per kilogram for nontested seed lots. To obtain viable seed, multiply values given by 100 and divide by the percent PLS of the seed lot to be planted. If one desires to seed a given number of viable seeds per linear meter, he can calculate seeding rate. For example, if 60 viable seed per meter of row is desired in 30-cm rows and the seed lot contains 220,000 PLS/kg, computation of seeding rate of PLS/ha would be: Planting rate in kg PLS =

Number of viable seed desired per row m X row m/ha Number of PLS kg

or Planting rate in kg PLS/ha = 6o 33’333 = 9.1 @/ha 220,000

The numbers of row meter per hectare for different row spacings are as follows: Row spacing (cm)

Row meter per hectare

15

30

45

60

90

66,666

33,333

22,222

16,666

11,111

Seeding rates for forage legumes vary from region to region and within regions depending upon seeding site condition. More seeds are sown than number of plants needed. In humid regions 70% emergence is considered excellent with good seeding techniques (Tesar and Jackobs, 1972) and the seedlings surviving the first year are 40 to 50% of the seed sown. A seeding rate of 11.2 kg/ha which was reported as average for the humid northeastern states in 1962 gave 538 seeds per square meter. From this seed 215 to 269 plants per square meter survived the first year. Jackobs and Miller (1970) have shown that this number will give maximum yield in the first harvest year in Illinois. They also report that 60 to 78 plants per square meter is adequate for maximum yield in subsequent years. In Montana 7.8 kg/ha of alfalfa provides adequate stands under irrigation.

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TABLE I Seed per Meter of Row for Some Common Forage Legumes Seeded at a Seeding Rate of 1 Kg/Ha at Different Row Spacings Row spacing in cm SDecies

Common name

Seeds/kg

15

30

90

60 ~

Astragalus cicer L. Coronilla varia L. Lespedeza cuneata Don. Lespedeza stipulacea Maxim. Lespedeza striata Hook Lk Am. Medicago sativa L. Melilotus alba Desr. Melilotus officinalis Lam. Lotus cornidatus ..I Onobrychis viciifolia Scop. Trifolium fragiferum L. Trifoliurn hybridum L. Trifolium pratense L. Trifolium repens L. Trifolium subterraneum L. Vicia sativa L.

Cicer milk vetch 280,950 4.2 Crown vetch 242,281 3.6 Sericea lespedeza 772,574 11.6 Korean lespedeza 496,654 7.4 419,298 6.3 Common lespedeza Alfalfa 4 4 1,472 6.6 White sweet clover 513,952 8.6 Yellow sweet clover 573,952 8.6 Birdsfoot trefoil 827,757 12.4 Sainfoin 66,220 1.0 Strawberry clover 662,208 9.9 Alsike clover 1,545,152 23.2 Red clover 607,023 9.1 White clover 1,765,887 26.4 Subterranean clover 143,471 2.2 Common vetch 15,450 0.2

8.4 16.8 25.3 7.3 14.5 21.6 23.2 46.3 69.6 14.9 29.8 44.4 12.6 25.2 37.8 13.2 26.5 39.6 17.2 34.4 51.6 17.2 34.4 51.6 24.8 49.6 74.4 4.0 2.0 6.0 19.9 39.7 59.4 46.4 92.7 139.2 18.2 36.4 54.6 53.0 105.9 158.4 4.4 8.8 13.2 0.8 1.2 0.4

C. DRILL CALIBRATION

Dnlls may be calibrated by weighlng the seed delivered over a given area or by converting kilograms of seed per hectare to number of seeds per row meter and then counting the number of seeds delivered per meter of row. The latter method is the easiest. Run the drill over hard ground or a canvas tarp and count the seed per meter of row. Adjust drill setting until desired number is obtained. Table I gives the number of seed per meter of row of a number of legumes at a 1 kg/ha seeding rate and at different row spacings. To obtain number of seeds per row meter multiply number of kilograms of PLS to be seeded by the value given for the row spacing to be used. For example, at a 10-kg PLS rate of seeding for alfalfa seeded in 15-cm row spacing, 66 seeds should be seeded per meter of row. For calibrating drills for seeding mixtures, mix the grass and legume in proper proportion and then count the number of seed of the legume per meter. Grass seed will automatically be seeded in the right proportion. Legumes and grasses establish best when seeded in alternate rows where they don’t compete against each other in early stages of development. Blocking off every other feed in the grain box for grass seed and alternate feeds in the legume box is an easy method of alternate row seedings. One-half-kilogram cloth bags filed with sand effectively block feeds.

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D. SEEDING

Forage legume seed may be broadcast or drilled. Broadcast seedings are seldom successful in the arid regions of the West but sometimes are successful in humid areas. Best results are obtained if seed is broadcast in early spring when the soil surface is cracked from freezing and thawing. Broadcast seed should be covered by harrowing lightly. Cultipacker seeders are used extensively for legume seedings. They consist of two corrugated rollers. The first roller firms the soil and leaves a small groove up to 2.5 cm deep. Seed, metered from a drill box, is broadcast into the grooves. The second roller covers the seed and firms the soil around it. Grain drills with a legume seed box do an excellent job of seeding, provided that the seedbed is firm and/or depth bands are used. Depth bands can be made locally or purchased from equipment dealers. The single disk drill is excellent for seeding hard and brushy seedbeds. Double disk drills are better on stubble or well-prepared seedbeds. Deep furrow drills place the seed in the bottom of a furrow, but it is covered at the normal seeding depth. These drills are effective in arid regions where snow and rainfall tend to concentrate in the bottom of the furrow. A danger of deep furrow planting is that in loose or erodable soils the furrows may fill in and cover the seed too deeply. The most reliable seeding method, where soil fertility is a problem, is band seeding. With this method, special drills place fertilizer in a band 3-6 cm deep. Seed is then drilled directly over the fertilizer band at a depth of 0.5 to 1.5 cm and the soil is firmed over the seed with press wheels. Press wheels are an asset to any drill when seeding legumes.

E. SOD SEEDING

Sod seeding has increased with the improvement of seeding equipment and the development of herbicides to control competition. Seeding success with no-till equipment is dependent upon: (1) reduction of competition, ( 2 ) slicing the sod for seed placement, (3) placement of seed at the proper depth, (4) firming soil over the seed, and ( 5 ) adequate moisture and fertility for good germination and growth. In Maryland, an offset concave disk placed between the leading straight coulter and spearpoint opener of a commercial grassland drill has consistently given good stands of birdsfoot trefoil and crown vetch. Deere and Co. is marketing a Power-Till seeder developed by the University of Kentucky (Ackley, 1975). This machine has sawlike cutter wheels which cut through surface residue and existing sod to open up a precise seed slot in the soil surface. Cutter wheels

TABLE I1 Seeding and Seeding Year Management Chart' Time (1) Prior to seeding

Prior to seeding

Operation

Select quality seed of To help insure good stands of adapted recommended varieties free productive species from weeds Inoculate legume seed with proper To provide symbiotic bacteria for nitrogen fixation bacteria, if needed

Prior to seeding

Level field if gravity irrigation is to be used Prepare a firm seedbed

Prior to seeding

Provide adequate fertility

Prior to seeding

(2) At seeding

Purpose

Seed at recommended depth

(3) Immediately after seeding Frequent observation to note soil crusting prior to emergence; if crusting occurs, go over land with light cultipacker

To insure uniform distribution of irrigation water To bring seed in close contact with moisture and nutrients; prolongs moisture retention for germination; to help control depth of seeding To stimulate development, decrease competition

Results Pastures with highly adapted species; weed control Production of nitrogen by legumes for use by grass; increased productivity; cheap source of nitrogen Increased productivity Good uniform stands; rapid emergence

Good stands and maximum seedling growth

To allow seed to emerge with energy available

Uniform emergence and good stands

To break up soil crust to allow emergence

Better emergence and stands

~~

(continued)

TABLE I1 (continued) Time (4) Dunng establishment

Operation

Purpose

Results

Frequent check of soil moisture; if To provide adequate water for seedling Rapid seedling development; dry, apply enough water to wet growth seedling root zone To increase photosynthetic activity of Vigorous seedlings, good stands; Frequent observation of weed higher yields in fust and competition; if weed competition seedlings strong, mow with guards set high or subsequent production years spray with proper herbicide

Cauti0ns:

(5) Do not seed with companion crop in close row spacing; if used, space companion crop in 18- to 21-inch Less competition to forage

rows. Use the same drill setting as normal for companion crop but plug 1/2 or 2/3 of spouts to obtain row spacing.

seedlings; increases chances for seeding success; increases forage yields

(6) Avoid grazing until late fall of seeding year.

Better establishment. Less winter injury; more productive stands

(7) Gear all management operations to meet needs of the forage seeding.

Seeding success

‘After Cooper et QL (1973).

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137

rotate in the direction of travel at 730 rpm, leave a slot 1.3 to 2.0 cm wide, and deposit loose soil in the slot. Seed is metered into the seed slot and firmed with packer wheels. A sprayer attachment then sprays a narrow band of herbicide, approximately 10 cm wide over the slot area to retard plant competition until seedlings have sufficient growth t o be competitive.

V I I I . Seeding Management Practices

Once legumes are seeded, periodic checking of the new seeding can pay dividends. Soil crusts that form before emergence may be broken up with a light cultipacker. Weed competition following emergence may be controlled with chemicals such as 2-4-D-B or by clipping just above the young seedling. Management practices for seeding and establishing a new legume seeding are presented in Table 11. Close adherence to these management principles will greatly increase the chances of seeding success. Aside from improving the vigor of legume seedlings, improvement of establishment practices is the major means of increasing seeding success. The often recommended firm seedbed needed for good establishment is seldom obtained in farm practice. Likewise, depth regulator bands which insure placement of seed at desired depth are too seldom used. There is a major need for seeding equipment designed specifically for seeding grasses and legumes. This equipment should insure the placement of the seed at the proper depth in a firm seedbed. It should also provide for simultaneous seeding of two species in alternate rows at different depths and should provide for band placement of fertilizer below seed. Proper application of the present state of knowledge concerning growth of the legume seedling should obtain successful establishment of legumes.

REFERENCES

Ackley, I. W. 1975. Proc. N o Tillage Forage Symp., Faucett Cent. Tomorrow, Columbus, Ohio pp. 53-71. Anderson, S. R. 1955. Agron. J. 41,483-487. Barton, L. V. 1947. Contrib. Boyce Thompson Inst. 14, 355-362. Beveridge, J . L., and Wilsie, C. P. 1959. Agron. J. 51, 731-734. Black, J. N. 1955. Aust. J. Agric. Res. 6 , 203. Black, J. N. 1956. Aust. J. Agric. Res. 7, 98-109. Black, J. N. 1957. Aust. J. Agric. Res. 8, 1-14. Black, J. N. 1959. Herb. Abstr. 29, 235-241. Blaser, R. E., Taylor, T., Griffith, W., and Skrdla, W. 1956. Agron. J. 48, 1 4 . Brant, R. E., McKee, G. W., and Cleveland, R. W. 1971. Crop Sci. 11, 1-6.

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Carleton, A. E., and Cooper, C. S. 1972. Crop Sci. 12, 183-186. Carleton, A. E., Wiesner, L. E., Dubbs, A. L., and Roath, C. W. 1967. Mont., Agric. Exp. Stn., Bull. 614. Carleton, A. E., Cooper, C. S., and Wiesner, L. E. 1968. Agron. J. 60, 81-84. Carleton, A. E., Austin, R. D., Stroh, J . R., Wiesner, L. E., and Sheetz, J. G. 1971. Mont., Agric. Exp. Stn., Bull. 655. Cooper, C. S. 1957. Agron, J. 49,473-477. Cooper, C. S . 1966. Crop Sci. 6,63-66. Cooper, C. S. 1967. Crop Sci, 7, 176-178. Cooper, C. S., and Ferguson, H. 1964. Agron. J. 5 6 , 6 3 4 4 . Cooper, C. S., and Fransen, S. C. 1974. Crop Sci. 14,732-735. Cooper, C. S., and MacDonald, P. W. 1970. Crop Sci. 10, 136-139. Cooper, C. S., Baldridge, D. E., and Roath, C. W. 1973. Mont., Agric. Exp. Sm., Bull. 622. Derwyn, R., Whalley, B., McKell, C. M., and Green, L. R. 1966. Crop Sci. 6, 147-150. Dotzenko, A. D., Cooper, C. S., Dobrenz, A. K., Laude, H. M., Massengale, M. A., and Feltner, K. C. 1967. Colo., Agric. Exp. Stn., Tech. Bull, 97. Draper, A. D., and Wilsie, C. P. 1965. Crop Sci. 5 , 3 13-3 1 5. Erickson, L. C. 1946. J. Am. SOC.Agron. 38,964-973. Fransen, S . C., and Cooper, C. S. 1976. Crop Sci. 16,434-437. Gist, G. R., and Mott, G. 0. 1958. Agron. J. 50, 583-586. Geiger, R. 1950. “The Climate Near the Ground” (M. N. Stewart, trans].) 4 8 2 pp. Harvard Univ. Press, Cambridge, Massachusetts. (“Das Klima der Bodennahen Luftschicht,” 2nd German Ed.) Gunn, C. R. 1972. In “Alfalfa Science and Technology” (C. H. Hanson, ed.), Agronomy Monograph, Vol. 15, pp, 677-686. Am. SOC. Agron., Madison, Wisconsin. Hensen, P. R., and Tayman, L. A. 1961. Crop Sci. 1, 306. Hoagland, D. R., and Broyer, T. C. 1936. Plant Physiol. 11,471-507. Jackobs, J. A., and Miller, D. A. 1970. Agron. Abstr. ASA p. 80. Jensen, E. H., Frelich, J. R., and Gifford, R. 0. 1972. Agron. J. 6 4 , 6 3 5 6 3 9 . Kidd, F., and West, C. 1919. Ann. Appl. Biol. 5:112-142. Lin, C-S. 1963. Master’s Problem, Montana State Univ., Bozeman. Lovell, P., and Moore, K. 1971.J. Exp. Bot. 22, 153-162. McElgunn, J. D. 1973. Can. J. Plant Sci. 53, 797-800. McKee, G. W. 1962. Pa., Agric. Exp. Srn., BUD. 689. McKell, C. M., Wilson, A. W.,and Williams, W. A. 1962. Agron. J. 54,109-1 13. McWilliam, J. R., Clements, R. J., and Dowling, P. M. 1970. Aust. J. Agric. Res. 21, 19-32. Moore, R. P. 1 9 4 3 . 1 Am. Soc. Agron 35,370-381. Nielsen, K. F., Halsted, R. L., Maclean, A. J., Holmes, R. M., and Bourget, S. J. 1961. Proc. Int. Grad. Congr., 8th, Reading, Engl., I960 8, 287-292. Opik, H., and Simon, E. W. 1963. J. Exp. Bot. 14, 299-310. Opik, H., and Simon, E. W. 1966. J. Exp. Bot. 17,427-439. Peiffer, R. A., McKee, G. W., and Risius, M. L. 1972. Agron. J. 64, 770-774. Pritchett, W. L., and Nelson, L. B. 1951. Agron. J. 43, 173-177. Qualls, M., and Cooper, C. S. 1968. Crop Sci. 8, 758-760. Richards, S. J., Hagan, R. M., and McAlla, T. M. 1952. In “Soil Physical Conditions and Plant Grow&’(B. T. Shaw, ed.), Agronomy Monograph, Vol. 2, pp. 302-480. Academic Press, New York. Rincker, C. M. 1954. Agron. J. 46,247-250. Shirley, H. L. 1945.Bot. Rev. 11,497-532. Smoliak, S., Johnston, A., and Hanna, M. R. 1972. Can. J. Plant Sci. 52,757-762. Sprague, V. G. 1943. Soil Sci. Soc. Am., Proc. 8,287-294.

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Stapledon, R. G., and Wheeler, D. E. 1948. J. Br. Grassl. SOC. 3, 263-271. Stickler, F. C., and Wassom, C. E. 1963. Agron. J. 55, 78. Stitt, R. E. 1944. J. Am. Soc. Agron. 36,464-467. Tesar, M. B., and Jackobs, J. A. 1972. In “Alfalfa Science and Technology” (C. H. Hanson, ed.), Agronomy Monograph, Vol. 15, pp. 415-433. Am. SOC. Agron., Madison, Wisconsin. Townsend, C. E., and McCinnies, W. J. 1972. Agron. J. 64, 809-812. Twamley, B. E. 1967. Can. J. Plant Sci. 47,603609. Twamley, B. E. 1974. Crop Sci. 14, 87-90. Uhvits, R. 1946.Am. J. Bot. 33,278-285. Wallace, A. 1957. Soil Sci. 83,407-411. Wanner, H. 1948. Ber. Schweiz. Bot. Ges. 58, 123-130. Watson, D. J. 1952. Adu. Agron. 4, 101-145. Williams, W. A. 1956. Agron. J. 48,273-274. Young, J. A., Evans, R. A., and Kay, B. L. 1970. J. RangeManage. 23,98-103.

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YIELDS AND CULTURAL ENERGY REQUIREMENTS FOR CORN AND SOYBEANS WITH VARIOUS TILLAGE-PLANTING SYSTEMS

. .

. .

. .

C B Richey. D R Griffith. and S D Parsons Purdue Agricultural Experiment Station. Lafayette. Indiana

I. introduction

..................................................

I1. Tillage-Planting Systems . . . . . . . . . . . . . . . . . .

.....................

A . Definition of Tillage-Planting System ............................ B. High-Energy Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Moderate-Energy Systems ...................................... D Low-Energy Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Influence of Tillage-Planting System on Yields ........................ A Corn ...................................................... B. Soybeans .................................................. IV. Yield Factors Influenced by Tillage-Planting System .................... A . Early Planting ............................................... B . Soil Compaction ............................................. C. Weed Control ............................................... D . Fertilizer Placement .......................................... E . Moisture Conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Soil Erosion Prevention ....................................... G . Insect and Disease Control ..................................... V. Energy Requirements for Various Tillage-Planting Systems . . . . . . . . . . . . . . A . Operations in Low-, Medium-, and High-Draft Soils .................. B . Various Tillage-Planting Systems in Low-, Medium., and High-Draft Soils . C. Corn Tillage Savings .......................................... D . SoybeanTillage Savings ....................................... VI . Projecting Energy Savings with Reduced Tillage ....................... VII . Conclusions ................................................... References ....................................................

. .

I

.

141 143 143 143 145 146 147 147 154 157 157 159 162 163 165 166 169 169 169 171 171 178 178 180 180

Introduction

Tradition has it that the Pilgrims were introduced to corn by the Indians.The Indians used a low-energy tillage-planting system wherein they dug a hole. dropped in a fish for fertilizer. and then planted a hill of corn . As plows and mechanical planters became available. farmers were able to substitute animal power for manual labor and thus multiply their output per 14 1

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C. B. RICHEY ET AL.

man. The object of tillage was to prepare an adequate seedbed and to control weeds. A good job of plowng with complete inversion of the soil aided in both respects, There was limited time for secondary tillage operations but cultivation for weed control became increasingly important as the weed population multiplied. When row-crop tractors with efficient equipment became available, farmers had the capacity to perform more tillage operations than in the past, particularly on small farms in years with good spring weather. They were able to disc corn stalks before plowing to aid in complete coverage of trash and they could disk, roll, and drag until they had the fine, uniform seedbed they had always desired. They could also cultivate up to five times in check-rowed corn to control weeds. Although most corn belt farmers had reached this point in mechanization by 1940, the average corn yield in the United States for the period 1937-1941 was 1824 kg/ha (29 bu/A), only 183 kg/ha (2.9 bu/A) higher than for the period 1867-1871. During the intervening period the high yield was 1994 kgfhg (31.7 bu/A) in 1906 and the low was 994 kg/ha (15.8 bu/A) in 1934 (USDA Agricultural Statistics, 1900, 1942). Thus, increased tillage was not the answer, and only as hybrid seed and increased fertilization came into use did corn yields begin their rapid advance to present levels. One of the earliest projects to evaluate tractor-powered tillage methods for corn was started in Ohio in 1938 (Willard et d.,1956). In a cornwheat-hay rotation on fairly level fine-textured Miami-Brookston soil, sod ground was prepared for planting corn by: 1. Spring plowing and several diskings. 2. Spring plowing with a prairie-breaker bottom which smoothly inverted the sod strip, followed by a smoothing harrow or straight disk. 3. Rotary tillage. 4. Surface tillage with sweeps or disk to kill the sod. 5. Surface tillage only to kill sod. 6. Treatment 1 with the addition of 4.4 tonnes/ha (2 tons/A) straw mulch after the first cultivation. Over the 14-year period 1938-195 1, the yields of plowed treatments were not significantly different and averaged about 3400 kg/ha, (54 bu/A); rototilled, 2956 kg/ha (47 bu/A); treatment 4, 281 1 kg/ha (44.7 bu/A); and treatment 5, 2528 kg/ha (40.2 bu/A). Stand, sod regrowth, and weeds were problems in treatments 4 and 5 . It was stated that “The results show conclusively that there is no advantage for corn in more working of plowed land than is necessary to insure a good stand, and there are indications that such working may be detrimental.” The advent of chemical weed control made it possible to control weeds without tillage in many cases. This greatly increased tillage-planting options and brought us into the present era of experimentation to find the optimum

YIELDS AND REQUIREMENTS FOR CORN AND SOYBEANS

143

combination of tillage, planting, and weed control practices to give maximum yields at minimum cost with minimum soil erosion. The purpose of this chapter is to review the tillage system effect on yield for corn and soybean production in the United States corn belt, and to estimate cultural energy requirements for various systems and soil types, including energy requirements for herbicides but not for fertilizer, harvest, and drying.

II. Tillage-Planting Systems

A. DEFINITION OF TILLAGE-PLANTING SYSTEM

Since the condition of the soil dictates the type of planting which is necessary t o secure a good stand, tillage and planting must be considered together. The type of cultivator required and its effectiveness in controlling weeds also depends on the condition of the soil. Thus the tillage-planting system includes preplant tillage, planting, and weed control by cultivation, herbicides, or both. The tillage system also affects methods of fertilizer and insecticide application. The tillage-planting system encompasses the operations needed to produce the crop ready for harvest.

B. HIGH-ENERGY SYSTEMS

In these systems the soil is thoroughly loosened at least 12 cm (5 inches) deep, either by moldboard plow, chisel, or heavy-duty disk harrow. Current corn belt practice is to plow about 20 cm (8 inches) deep if power and soil condition permit. The average depth of chiseling is usually comparable to plowing depth, with the points penetrating the old plow sole. This is considered to be an advantage for chiseling because it improves water infiltration. Chiseling is also considered less likely to develop a “sole” or impenetrable layer than is plowing. Heavy-duty disk harrows with 61-cm (24-inch) diameter blades can cut up to 15 cm (6 inches) deep with a draft per unit of cross section tilled comparable to that for a chisel plow, or about 80%of that for a moldboard plow. They also cut and cover most of the residue. Plowing and chiseling are preferably done in the fall on level fine-textured soils which are not subject to winter erosion. Chiseling after corn leaves more residue on the surface than plowing and thus does not leave the soil as vulnerable to water and wind erosion as does plowing. Residue effects are minimal after soybeans. Corn stalks are often shredded or disked to avoid clogging, although high-

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ET AL.

clearance plows and chisels can often operate without stalk treatment. Some chisels use a gang of straight coulter-blades across the front to cut the stalks and prevent clogging. One chisel uses an opposed pair of disk gangs across the front with the angle of cut adjustment controlling the proportion of residue left on the surface for erosion control. Secondary tillage operations are performed in the spring and usually include an early disking to level the soil and a disking or field cultivation just ahead of the planter to lull weeds, break any crusting, and secure a finer, firm seedbed.' The disk may also be used to incorporate broadcast herbicides and insecticides where needed. In some areas special mulching tools have become popular for the final preplant operation. They consist of various combinations of spring teeth, rollers, spike teeth, and leveling blades to break up clods and obtain a very level, firm seedbed. Planting is helped by high-energy tillage because there is little surface residue, allowing the use of runner openers rather than disk openers, and the level seedbed facilitates uniform depth of seeding. Major plant nutrients can be applied in a variety of ways with high-energy systems. Phosphorus (P) and potassium (K) for corn are normally bulk spread before primary tillage or before the final secondary tillage operation. Volatile forms of N (NH,) can be knifed in separately or in combination with tillage operations. Nonvolatile forms of N can be broadcast on the surface or applied as starter fertilizer along with P and K by the planter. Insecticides are often band-applied by the planter and preemergence herbicides can also be banded or broadcast as spray or granules if they have not been previously applied. Planters of more than eight rows are sometimes not equipped with fertilizer or herbicide attachments in order to reduce lost time during planting. High-energy systems also facilitate mechanical cultivation because there is little surface residue to clog, and the soil is loose enough for shovels and sweeps to operate effectively. If the herbicide is effective no cultivation may be necessary. Many farmers plan to cultivate corn once and soybeans twice, with more cultivation being done if needed. In general, the high-energy systems provide a greater factor of safety than low-energy systems because extra operations can be done if needed. An extra The practice of wheel-track planting completely eliminates secondary tillage. The ground is plowed only a day or so ahead of planting and then the rows are planted in tractor and planter wheel tracks. In spite of good yield response this system has lost favor as acreages increased because of the economic handicap of having enough plowing capacity to keep ahead of today's large planters. It is also not compatible with incorporation of broadcast herbicides.

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preplant tillage operation can be done if a heavy rain causes crusting, or an extra cultivation if the herbicide is not effective. The high-energy systems also provide more options for fertilization and pest control.

C. MODERATE-ENERGY SYSTEMS

In these systems the soil is usually tilled less than 10 cm (4 inches) deep, with a total energy requirement for tillage and planting two-thirds or less of that with hgh-energy systems. Specialized equipment may be needed and there are fewer options for fertilization and pest control. The most popular moderate-energy system is disking with a conventional tandem disk, although surface residue makes necessary a disk-opener planter. One or two spring diskings often provide a good seedbed following soybeans, since there is little residue and the soil tends to be loose. More disking may be required following corn. Disking is often a wise choice when wet weather has delayed high-energy spring tillage, because deep tillage of wet soil can result in large hard clods if the weather turns dry. It also facilitates incorporation of fertilizer, herbicides, and insecticides. The ridge system developed in Iowa (Buchele et al., 1955) has moderate energy requirements. After the permanent ridges have been initially formed, they are maintained by cultivation. Stalks are shredded in the spring before planting and tend to settle in the furrows. A conventional planter is guided by large disks bearing against the ridge sides and plants on the old ridge top, disregarding the old stubs. A fall ridging system requiring somewhat more energy has been developed in Indiana (Richey e l al., 1973). Ridges are reshaped in the fall after harvest by a combined flail shredder and disk bedder. The stalk residue is picked up by the shredder, elevated over ridging disks located just behind the shredding rotor, and funneled into the open furrows between ridges. The ridges are smoothed in the early spring by a rolling cultivator when necessary, and planting is done directly on the ridge top in the mellow soil which was thrown u p in the fall. The planter is guided on the ridges by disks bearing against the ridge sides or by wide furrow-fitting tires on the planter transport wheels. Ridge systems provide a warmer seedbed than other tillage systems having surface residue, and the residue in the furrows helps t o control runoff and erosion. There is reduced inundation of seedlings in wet weather compared to flat planting. The fall furrow-mulch ridging system provides winter erosion protection and does not require cultivation to reshape the ridge. Fertilizer broadcast in the fall before ridging is concentrated in the ridge. Anhydrous ammonia can be knifed

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C. B. RICHEY ET AL.

into the furrows in the spring by using coulters to cut the trash ahead of the knives.

D. LOW-ENERGY SYSTEMS Strip or slot tillage of the seed row in combination with planting requires only about one-fourth the tractor energy required for high-energy systems, although herbicide costs may prevent an overall saving. The till-plant system, which was developed in Nebraska (Wittmuss and Lane, 1973) and is widely used in the western corn belt, requires minimum energy for planting but requires at least one ridge-shaping cultivation to maintain the ridges. In this system, a 25- to 35-cm (10- to 14-inch) wide flat sweep preceding the planter opener slices about 6 cm (2.5 inches) from the top of the ridge, pushing stalk residue and root clumps into the middles. Shredding the stalks ahead of the planter eases cultivation, although it is not necessary for planting. Till-planting evolved from an experimental design by Poynor (1950) and further development in Nebraska (Lane and Wittmuss, 1961). One till-planter forms a V-furrow in the exposed moist soil, drops in seed, presses it down with a narrow seed press wheel, and covers the seed with loose soil from two small disk coverers. Uniformity of depth and moisture content is good and the loose covering soil is not as likely to crust as when compacted by a large press wheel such as used on conventional planters. After planting, the row area is usually slightly lower than the loose material in the middles. Clearing the row area of residue aids soil warm-up, resulting in better germination and better early growth than with the no-till system where the residue is left in place. Rolling cultivators are often used because they do not clog easily, and the gangs can be tilted to rebuild the ridges. Disk cultivators are also popular. Herbicides may be broadcast at planting or banded since a cultivation wiIl be needed to rebuild ridges. If not cultivated the depressed row area causes increased stubble loss when harvesting soybeans. The “no-till” coulter system is the most popular lowenergy system and has had wide acceptance for corn in the southern corn belt and for doubre-cropping of soybeans after small grains. Coulters with flutes ranging from 6 cm (2 1/2 inches) down to about 1 cm (1/2 inch) wide are used to cut a path through the residue and loosen a slot in the soil for the planter opener. Since the pressure of the coulter tends to depress the soil, the planter press wheel usually has a rib in the center to aid in firming the soil over the seed. Corn stalks are usually shredded to obtain a uniformly distributed mulch. No residue preparation is necessary in the case of a chemically killed sod crop or soybean stubble.

YIELDS AND REQUIREMENTS FOR CORN AND SOYBEANS

147

Strip rotary tillers have been used to till strips about 20 cm (8 inches) wide and slightly deeper than planting depth. A conventional planter is trailed behind or unit planters are mounted on the tiller for a once-over operation. Stalk shredding can often be omitted because the residue in the row is well chopped. Energy requirements are very similar to the no-ti11 coulter system, as is plant growth. Cultivation is difficult with either the coulter or strip rotary system because of the lack of loose soil. Cultivators in general are not adapted to work in firm untilled soil, so herbicides must be relied upon for weed control. Bulk applications of P and K should be made before planting. Nitrogen may be applied in bulk as a nonpressure solution with or without herbicide. Anhydrous ammonia (NH,) may be knifed in, although a residue cutting coulter must precede the knife, and extra sealing wings may be needed on the knife to prevent loss because of the firmness of the soil. Starter fertilizer is desirable to help overcome the initially adverse environment. Ill. Influence of Tillage-Planting System on Yields

A. CORN

1, Indiana Experiments

An interdisciplinary project to compare various tillage planting systems for continuous corn was initiated in 1967 (Richey et al., 1973). The chemical and mechanical analysis of the soils is shown in Table I (Griffth et al., 1973). The results for northwestern Indiana are shown in Table 11. On the Tracy sandy loam (typic hapludalf) there was little difference between systems. In the 1967-1971 period, the till-plant system had significantly higher yields but this is thought to be primarily a result of the single cultivation needed t o build up a ridge for the following year. In subsequent years cultivation was found to also increase yields with the other tillage systems on this soil. On the heavier Runnymede loam (typic argiaquoll) fall plowing showed a 942 kg/ha (15 bu/A) advantage over spring plowing and more over the other treatments for 1969-1971. The no-till coulter system was at a considerable disadvantage, yielding substantially less than the other systems and almost one-third less than fall plowing in 1969-1971. The results for east central and southern Indiana are shown in Table 111. On the heavy Blount silt loam (aeric ochraqualf) and Pewamo clay loam (typic argiaquoll), conventional plowing was superior to the other treatments with disking, no-tiU, and rotary strip tillage decidedly inferior.

148

C. B. RICHEY ET AL.

TABLE I Chemical and Mechanical Analysis of Indiana Soils to 15 cm (6 inches) Depth, 1967' ~~

Soil type and location Tracy sandy loan (typic hapludalf) Northwestern Indiana Runnmede loam (typic argiaquoll) Northwestern Indiana Blount silt loam (aeric ochraqualf) East central Indiana Pewamo silty clay loam (typic argiaquoll) East central Indiana Bedford silt loam (typic fragiudult) South central Indiana

~

Total Total Total Organic KC sand silt clay matter Pb pH kg!ha (lb/A) kg/ha (lb/A) (%) (%) (%) (%)

6.1

113(101)

317(283)

59.36 38.93

2.64

1.41

6.3

104(93)

240(214)

47.48 44.79

7.73

2.99

6.7

lOl(90)

323(288)

20.67 58.64 20.67

1.74

6.5

226(202)

306(273)

14.07 47.40 38.50

3.28

6.4

64(57)

204(182)

10.02 83.75

1.71

6.22

'Griffith ef al. (1973). bMedium range for P 46-80 kg/ha (41-71 lb/A). CMediumrange for K 151-210 kg/ha (135-187 Ib/A).

On the Bedford silt loam (typic fragiadult) in southern Indiana, all systems averaged about the same for 1967-1971, except for the till-plant which again showed an increase possibly due to its cultivation, Residue on the surface appeared to provide a slight yield advantage. Moisture conserved in the summer may have overbalanced the lower soil temperature early in the season.

2. Ohio Experiments Studies comparing corn yields of conventional plow tillage with no-till coulter planting under several crop rotation combinations on several typical Ohio soils were conducted in the 1962-1973 period. The results are summarized in Table IV (van Doren et al., 1976). Plots were thinned to achieve a common plant population. No-till gave significantly higher yields than plowing under continuous corn and a corn-soybean rotation on Wooster silt loam but significantly lower under continuous corn on Hoytville silty clay loam.

TABLE I1 Corn Response to Tillage-Planting System, Northwestern Indiana Tracy sandy loam

1967-1971 Tillage-Planting Fall plow -conventionalc Spring plow-conventional Spring plow-wheel-track plant Fall chisel-conventionalc Fall disk-conventionalc Till-plant, one cultivationd Till-plant, no cultivation Rotary strip till and plant No-till coulter plant Fall ridge leaving residue in furrow

1969-1971

Standa

Yieldb

Standa

-

-

50.4 (20.4) 46.5 (18.8) 46.9 (19.0)

7846 (125) 7846 (125) 8223 (131)

49.2 (29.9) 49.9 (20.2) 44.5 (18.0) 50.9 (20.6)

8976 (143)

-

-

51.1 (20.7) 51.9 (21.0)

8160 (130) 7972 (127) -

-

1972-1973

Yieldb Standa

-

50.7 (20.5)

Runnymede loam

51.4 (20.8) 51.4 (20.8) 51.1 (20.7) 55.8 (22.6) -

%and 4 weeks after planting in 1000 plants/ha (1000 plants/A). bYield in hg/ha (bu/A). ‘In 1973 only the ridged plots were tilled in the fall. dNo cultivation for any system except till-plant as shown.

-

49.9 (20.2) -

Yieldb

-

1967-197 1 StandQ Yieldb

-

7124 49.9 (113.5) (202) 45.7 (18.5) 46.7 7482 48.2 (18.9) (119.2) (19.5) 44.5 6779 (19.0) (108) 52.9 7934 54.4 (21.4) (126.4) (22.0) 52.6 7589 (21.3) (120.9) 51.1 (20.7) 50.9 7733 52.4 (20.6) (123.2) (21.2) 51.9 7558 (21.0) (120.4)

8662 (138) 8223 (131) 8286 (132) -

1969-1 97 1 Standa

51.4 (20.8) 53.4 (21.6) -

Standa

Yieldb

51.4 (20.8)

55.1 (22.3) 50.2 (20.3) 43.7 (17.7) 49.9 (20.2) -

8574 54.1 (136.6) (21.9) 8035 (128) 7281 (116) -

Yieldb

1972-1974

-

53.4 (21.6)

8913 (142)

50.2 (20.3) 52.8 (21.4)

8072 (129)

TABLE 111 Corn Response to Tillageplanting System, East Central and Southern Indiana Blount silt loam

1968-1 97 1 Tillage-Plan ting Fall plow-conventionalc Spring plow-conventional Spring plow-wheel track plant c v,

Fall chisel-conventionalc

0

Fall disk-conventionalc Till-plant, one cultivationd Rotary strip till and plant No-till coulter plant Fall ridge with residue in furrow'

Standa

51.6 (20.9) 50.2 (20.3) 50.2 (20.3) 45.5 (18.4) -

50.2 (20.3) 43.0 (17.4) 51.4 (20.8) -

Yieldb

Pewamo silty clay loam

1972-1973 Standa

Yieldb

42.5 (17.2) -

41.8 (16.9) 56.8 (23.0) 46.9 (19.0) 42.0 (17.0) 49.2 (19.9)

aStand 4 weeks after planting in 1000 plants/ha (1000 plants/A). bYield in kg/ha (bu/A). CIn1973 all tillage was done in the spring. dNo cultivation for any system except till-plant, as shown.

196 8-1 97 1

Bedford silt loam

1974-1 97 5

Standa

Yieldb Standa

46.7 (18.9) 48.7 (19.7) 41.3 (17.1) 43.0 (17.4)

55.1 (22.3)

1967-1971

Yieldb

Standa

5856 (933)

-

51.9 (21.0) 45.7 (18.5) 5166 48.4 (82.3) (19.6) -

54.1 (21.9) -

-

45.2 (18.3) 42.0 (17.0)

-

54.4 (22.0)

5097 (81.2)

53.6 (21.7) 45.7 (13.5) 46.7 (18.9)

-

Yieldb

1972-1973 Standa

Yieldb

50.2 (20.3) 46.9 (19.0) 41.8 (16.9) 46.7 (18.9) 41.7 (19.3) 49.4 (20.0)

7783 (124) 7093 (113) 7532 (120) -

TABLE IV Average Corn Yield within Each of Four Ohio Locations, Three Rotations, Two Time Periods, and Two Sets of Tillage Treatmentsa Remaining years (1969-1973)

Years 2 4 (1963-1968)

Soil type

r

cn

r

Crop rotation

No-till kg/ha (bu/A)

Plow kg/ha (bu/a)

No-till Probabilityb kg/ha (bu/A)

Plow kg/ha Cbu/A) Probabilityb

Wooster silt loam (typic fragiudalo

Continuous corn Corn-soybeans Corn-oats-ha y

7060 (112) 6470 (103) 6880 (110)

6370 (101) 5900 (94) 6850 (109)

0.001 0.015 ND

9400 (150) 9480 (151) 10450(166)

8420 (134) 8720 (139) 9720 (155)

<0.001 0.03 0.01

Crosby silt loam (aeric ochraqualf)

Continuous corn Cornsoybeans Corn-oats-hay

6120 (97) 6410 (102) 7000 (112)

6180 (98) 6510 (104) 6720 (107)

ND ND ND

8620 (137) 8290 (132) 9730 (155)

8290 (132) 9020 (144) 9340 (149)

ND 0.07 ND

Hoytville silty clay loam (mollic ochraqualf)

Continuous corn Cornsoybeans Corn-oats-hay

5720 (91) 6720 (107) 6830 (109)

6490 (103) 7000 (1 12) 6800 (108)

<0.001 ND ND

6820 (109) 7920 (126) 8180 (130)

8000 (1 27) 8260 (132) 8390 (134)

<0.001

Toledo clay (mollic haplaquept)

Continuous corn Cornsoybeans Corn-oats-hay

5060 (81) 5350 (85) 5080 (81)

5250 (84) 5560 (89) 5270 (84)

ND ND ND

6520 (104) 6930 (110) 7220 (115)

6340 (101) 7000 (112) 7550 (120)

ND ND ND

W a n Doren et al. (1976). bProbability level at which the yield difference between pairs of tillage treatments is equal to zero. ND means no difference (p > 0.20).

ND ND

152

C. B. RICHEY ET AL.

Effects of plowing, disking, cultivating, and no-tillage on corn yield and mulch cover were investigated from 1962 to 1971 (Van Doren and Triplett, 1973). It was reported that: “Mulch cover produced three times as great a yield effect as any other single variable, with the effect (kg/ha) = 36.2 M - 0.0021 8 MS, where M is percent of the soil surface covered with mulch and S is yield in kg/ha of the plowed plus cultivated treatment. Tillage variables increased yield in order of cultivation > plow > disk, and results are expressed in terms of mulch cover required to produce an equal yield effect. Most tillage and mulch effects were additive.” It was postulated that soils could be classified as follows: A. Soils in which tillage and mulch effects on yield primarily result from water conservation. . . . B. Soils in which certain types of tillage can overcome mechanical impedance limitations to root and water penetration. . . . C. Soils and climates in which mulch effects on soil temperatures influence corn yield. . . . D. Soils in which excess water occurs for substantial periods, probably causing aeration problems for the growing crop. . . .”

3. Illinois Experiments A project was initiated in 1971 to study the effects of different tillage treatments and the resulting amount of corn stalk residue on the soil surface on corn yield and soil loss. The results are shown in Table V (Oschwald and Siemens, 1976). The disk-chisel had a pair of adjustable-angle disk gangs mounted ahead of the chisels. The coulter-chisel had a gang of coulters across the front to cut stalks. Tillage systems had little yield impact on Catlin silt loam, a soil with good drainage. The stands were about the same with all treatments, and weed control was excellent. Corn and soybean growth tended to be slightly slower with the no-till coulter treatment, but less so than on soils with restricted drainage. The Flanagan silt loam has poor natural drainage. Planting was performed as early as possible, even when some treatments were too wet. In general, yields were lowest with spring plowing and spring disking, where there was the most manipulation of wet soil in the spring. Wet soil in 1973 resulted in severe crusting and poor stands with all treatments except no-till coulter planting and fall plowing.

4. Nebraska Experiments In 1960, 16 comparison tests were made in 15 counties. Each test compared 1 ha (2.5 A) of plowed and conventionally planted corn with 1 ha (2.5 A) of

TABLE V Corn Yields and Soil Erosiona with Various Tillage Systems in Central Illinoisb Catlin silt loam (typic argiudoll)

Tillage-planting system Chop stalks, fall plow, disk, plant Spring plow, disk, plant Fall disk-chisel, disk-chisel (sweeps), plant Fall coulter-chisel, field cultivate, plant Chop stalks, fall chisel, disk, plant Chop stalks, plant with no-till coulter Spring disk twice, plant

Flanigan silt loam (aquic argiudoll)

Sediment loss 1000 kg/ha (lb/A)

1972, 1973, 1975 Average kg/ha (bu/A)

1972-1975 Average kg/ha (bu/A)

14.7 (16.5)

9102 (145)

-

-

4.0 (4.5) 3.5 (3.9) 5.7 (6.4) 2.5 (2.8) 2.9 (3.3)

8976 (143) 8976 (143) 9164 (146) 8537 (136) 8913 (142)

9290 (148) 8412 (134) 8913 (142) 8851 (141) 8976 (143) 9102 (145) 8662 (138)

aWater applied at 6.3 cm (2.5 inches) per hour for 4 hours by rainfall simulator after planting corn in 1973. bOschwald and Siemens (1 976).

154

C.

B. RICHEY ET AL.

till-planted corn (Wittmuss et al., 1971). The till-planted corn averaged 7470 kg/ha (1 19 bu/A) compared with 1744 kg/ha (1 17 bu/A) for the conventional corn, ranging from a difference of 1381 kglha (22 bu/A) favoring till-plant to a difference of 1130 kg/ha (18 bu/A) favoring conventional. Since 1960 till-planting has become common in the western corn belt, displacing the traditional lister system as well as the conventional plowing system.

B. SOYBEANS

1. Indiana Experiments A project to compare four tillage-planting systems on a continuous corn, continuous soybeans, and a corn-soybean rotation was initiated in 1974 near Lafayette on Chalmers silty clay loam (typic argiaquoll). The yields results for soybeans are shown for the first 2 years, 1975 and 1976, in Table VI. Soybean growth in corn residue was slower, like corn growth, but differences were smaller than for corn. There was little growth difference when planted in soybean residue. Slow growth in the 1975 ridged plots resulted from delayed germination, a consequence of shallow planting followed by dry weather. Yield differences were small following corn but no-till coulter yields were reduced substantially in the bean residue. Soybean yields for all treatments were consistently higher for beans after corn than for beans after beans, although early growth favored the latter in 1975. Visual observations indicated no difference in disease symptoms throughout the plot area.

2. nlinois Experiments Tillage experiments similar to those described previously for corn were also conducted for soybeans following corn (Siemens and Oschwald, 1975). The results are shown in Table VII. The yield differences were not statistically different.

3. Ohio Experiments Projected average soybean yields in a corn-soybean rotation with different tiUage systems for several Ohio soils are shown in Table VIII (Bone et al., 1976). It would appear that soybean yields are less affected by tillage variations than corn, showing only small differences with equal weed control. Tillage effectiveness in controlling weeds is a major factor.

TABLE VI Soybean Response to Tillage-Planting System and Previous Crop in West Central Indiana on Chalmers Silty Clay Loam (Typic Argiaquoll) ~~

1975 (Planted 5/6 and 7)

Tillage-planting system

Fall plow-conventional Fall chisel-conventional Fall ridge leaving residue in f u r r o d Nc-till coulter plant Fall plow-conventional Fall chisel-conventional FaJI ridge leaving residue in f u r r o d No-till coulter plant

1976 (Planted 5/10 and 11)

Mean

Height at 8 weeks Yield Height at 8 weeks Yield Yield Previous Height at 8 weeks cm (inches) cm (inches) kg/ha (bu/A) crop cm (inches) kg/ha (bu/A) @/ha (bu/A) Corn Corn Corn Corn Soybeans Soybeans Soybeans Soybeans

56.6 (22.3) 54.9 (21.6) 43.2 (17.0) 49.3 (19.4) 58.7 (23.1) 58.2 (22.9) 53.1 (20.9) 58.4 (23.0)

3793 (56.4) 3874 (57.6) 3356 (49.9) 3766 (56.0) 3544 (52.7) 3510 (52.2) 3302 (49.1) 3215 (47.8)

46.7 (18.4) 44.3 (17.4) 48.1 (18.9) 41.0 (16.1) 44.8 (17.6) 43.7 (17.2) 47.0 (18.5) 43.9 (17.3)

3657 (54.4) 3409 (50.7) 3425 (50.9) 3250 (48.3) 3225 (48.0) 3059 (45.5) 3093 (46.0) 2781 (41.4)

51.7 (20.3) 49.6 (19.5) 45.7 (18.0) 45.2 (17.8) 51.8 (20.4) 5 1.o (20.0) 50.1 (19.7) 51.2 (20.1)

%advertent shallow planting in 1975 resulted in reduced and delayed germination, impairing stand, early growth, and yield.

3725 (55.4) 3642 (54.1) 3391 (50.4) 3508 (52.2) 3385 (50.3) 3285 (48.8) 3198 (47.5) 2998 (44.6)

TABLE VII Soybeans Yields (After Corn) and Weed Weights with Various Tilling Systems in Central Illinoisa Flanagan silt loam (aquic argiudoll) Catlin silt loam (typic argiudoll) 1974

Soybeans kg/ha (bu/A)

Tillage-planting system

Weedsb kg/ha (lb/A)

Soybeans kg/ha (bu/A)

1973'

1974

Mean

Chop stalks, fall plow, disk, plant Fall disk-chisel, disk-chisel (sweeps), plant Fall coulter-chisel, field cultivate, plant Chop stalks, fall chisel, disk, plant Chop stalks, plant with no-till coulter Spring disk twice, plant

7.4 (6.6) 135.8 (121.2) 74.9 (66.9) 276.7 (246.9) 60.1 (53.6) 209.3 (186.7)

2892 (43) 2690 (40) 2825 (42) 2757 (41) 2421 (36) 2623 (39)

2576 (38.3) 2784 (41.4)

3067 (45.6) 3080 (45.8) -

2825 (42.0) 2934 (43.6)

-

3013 (44.8) 3013 (44.8)

-

2979 (44.3) 2898 (43.1)

2912 (43.3) 3134 (46.6)

-

~~

%emens and Oschwald (1975). bMeasured in mid-September. Weeds were a mixture of green foxtail (Setaria viridis 1. Beauv.), fall panicum (Panicum dichoromiflorum Q Michx.), and common milkweed (Asclepias S J T ~ ~ ~ CL.). 'Corn grown the previous year was harvested late and losses were high. All tillage operations were done in the spring. Volunteer corn was a serious problem in the fall-plow treatment especially and may have reduced yields.

YIELDS AND REQUIREMENTS FOR CORN AND SOYBEANS

157

TABLE VIII Soybean Yields after Corn with Various Tillage-Planting Systems on Several Ohio Soils, Based on Experimental Results’

Soil type Wooster silt loam (typic fragiudalf) Rossmoyne silt loam (aquic fragiudalf) Crosby silt loam (aeric ochraqualf) Brookston silt loam (typic argiaquoll) Hoytville silty clay loam (mollic ochraqualf)

Conventional tillage Chop stalks, fall plow, disk twice, plant k d h a (bu/A)

Minimum tillage Fall chisel, disk, plant kdha W A )

No-till Chop stalks, plant with no-till coulter kg/ha (bu/A)

2421 (36)

-

2219 (33)

3228 (48)

2959 (44)

2757 (41)

3430 (51)

3228 (48)

3699 (55)

3491 (52)

3430 (51)

3228 (48)

2556 (38)

-

2623 (39)

“From Bone et al. (1976). IV. Yield Factors Influenced by Tillage-Planting System

Although average yields over several years may show little difference between some systems, there are often significant differences in individual years, depending primarily on climatic conditions. These yield differences are best explained by examining the major factors controlling yields and the influence of the tillage-planting system on these factors. A. EARLY PLANTING

Corn yields in the central corn belt have been found to decrease approximately 63 kg/ha (1 bu/A) per day (Barber, 1965; Pendleton and Egli, 1969; Bone et al., 1976) after May 10 and even more after May 24. In order to have most of the crop planted by the start of the penalty period it is necessary for a farmer with a large acreage to start planting as early as he can get an adequate and healthy stand. The primary requirements for successful planting are: 1 . Soil dry enough to permit proper planter functions. 2. Adequate warmth and moisture for germination and early growth. 3. Minimum crusting impedance to emergence. Soil does not warm until it has dried. Tillage treatment has little influence on soil temperature and plant growth in a dry spring but it can make a substantial difference in a wet spring.

TABLE IX Mean Daily Maximum Soil Temperature in Row at 10 cm (4 inches) Depth for the 8 Weeks Following Planting Corn in Indiana, in "C ( O F ) Chalmers Tracy silt loam Runnymede loam Blount silt loam Pewamo silty clay loam Bedford silt loam silty clay loam Tillage-planting system

Fall plow-conventional Spring plow-conventional

Fall chisel-conventional Till-plant No-till coulter plant Fall ridge with residue in furrow

19691971

19721973

19691970

19721974

19691970

19721973

19691970

1972,1974, 1975

23.3 (73.9) 23.3 (73.9) 20.6 (69.1) 21.6 (70.9) 19.1 (66.4)

24.7 (76.4)

21.9 (71.4) 21.7 (71.1) 19.6 (67.3) 20.8 (69.4) 18.2 (64.8)

25.2 (77.4) -

24.6 (76.3) 24.3 (75.2) 22.4 (72.4) 23.4 (74.1) 22.1 (71.7)

23.2 (73.8)

24.5 (76.2) 24.3 (75.0) 22.4 (72.4) 23.5 (74.3) 23.2 (73.7)

23.2 (73.8) -

-

-

23.7 (74.7) 21.4 (70.6) 25.5 (77.9)

-

22.1 (71.8) 25.2 (77.4)

-

-

22.4 (72.4) -

21.9 (71.4) 23.1 (73.6)

-

22.7 (72.9) -

23.1 (73.6)

19691970

19721973

19751976 24.6 (76.3)

25.6 (78.1) 24.2 (75.6) 25.1 (77.2) 23.4 (74.2) -

26.4 (79.6) 25.2 (77.4)

24.2 (75.6)

24.1 (75.4) 25.2 (77.4)

22.4 (72.3) 24.2 (75.6)

-

YIELDS AND REQUIREMENTS FOR CORN AND SOYBEANS

159

Mean maximum daily soil temperatures in the row at a depth of 10 cm (4 inches) during the 8 weeks following planting in various Indiana soils are shown in Table IX. Fall plowing with conventional spring tillage showed the highest soil temperatures. The loosened soil dried well in the spring and there was little surface residue to shade the soil. Chiseling was slightly lower due to the residue left on the surface. Ridges with residue mostly in the furrow averaged slightly warmer than chiseling but below plowing, perhaps because soil in the ridges was not loose, except for the reshaped soil thrown up in the fall. With no-till planting in shredded corn stalks, the soil was not loosened t o aid drying and the residue shaded most of the surface, resulting in temperatures around 2.5'C (4.5'F) below plowing treatments. This definitely slowed early growth in central and northern Indiana. Crusting impedance to emergence can result from a heavy rainfall after planting on vulnerable soils. A finely worked surface and soil firming over the seed increases the tendency to crust. The till-planter technique of pressing the seed into firm moist soil and covering it with uncompacted loose soil results in high germination and minimum crusting. In 1972 a heavy rain immediately following planting caused severe crusting on the Bfount and Pewamo soils in eastern Indiana. All stands were severely reduced from the 59,300 kernels per hectare (24,000 kernels per acre) planted but the till-planter plots and the ridge plots (also planted with the till-planter) averaged 39,000 plants per hectare (15,800 plants per acre) compared with an average of 27,675 plants per hectare (1 1,200 plants per acre) for all other plots.

B. SOIL COMPACTION

Soil managed for minimum compaction will have increased water infiltration, reduced erosion, and increased aeration and will allow greater root penetration. It has often been difficult to obtain direct effects of compaction on yield because plants are quite adaptable. A plant with a small root system having access to adequate water and nutrients may yield as much as one with a large root system. Experiments in Illinois (Bateman, 1962) compared medium compaction by plowing and two diskings with several treatments designed to produce high compaction: a. Plowed, six tandem diskings. b. Plowed and two diskings, also compacted by two passes of tractor tires over all the plot after planting and between rows after the last cultivation.

160

C. B. RICHEY ET AL.

c. Two pass tire compaction in bottom of each furrow and on the land, plowed, two diskings. The 1960 and 1961 results for the means of the high compaction treatments versus medium compaction are shown in Table X for two soils. It was concluded that: Corn growth may be expected to be retarded when the air voids at field capacity moisture are near the 10% value. Many results have indicated that tractor tire traffic can reduce air voids to the critical value of 10% or less in many soil types, and the low values are easier to develop at the higher soil moisture contents. The Drummer silty clay loam appeared to be more susceptible than the Thorp silt loam to compaction. Important factors in the difference are the higher clay content of Drummer and its normally higher moisture when the soil is tilled.

Experiments in New York (Free and Bay, 1964) found that nine diskings and harrowings compared to one reduced corn yields only 5.2 hl/ha (6 bu/A) in the fourth year of treatments but the difference increased t o 19.1 hl/ha (22 bu/A) the fifth year and 27.8 hl/ha (32 bu/A) in the sixth year. Winter freezing and thawing ameliorates compaction, at least down to plowing depth, in most of the corn belt. In the warmer southern states compaction is more serious. Research in Alabama (Dumas et al., 1973) found that plow sole compaction and tractor wheel compaction between the rows left about 25% of the soil profile to 46 cm (18 inches) depth loose enough for good cotton root development by midsummer. With 46 cm (18 inches) deep tillage and controlled traffic where all operations were performed with a 3-m (120-inch) wheel tread tractor running on untilled paths, 55% of the soil profile was available for root penetration away from the wheel tracks and 44% including the wheel tracks. The deeptilled controlled-traffic plots yielded up to 37% more cotton than the conventional plots. Inasmuch as wheels are the primary source of compaction, it is possible to reduce compaction by (a) reducing tractor and machine weight, (b) reducing the number of trips, and (c) reducing the area compacted by practicing controlled traffic. Moderate- and lowenergy tillage systems should reduce compaction because lower-powered lighter tractors can be used and fewer trips are made across the field. Also, there is less disturbance of the favorable soil structure which develops under grassland with its continuous macropores, due to root and worm channels which facilitate infiltration (Van Doren et al., 1976; Baeumer and Bakermans, 1973). Controlled traffic is not presently practical with plow or chisel systems where the old rows are obliterated. The ridge and no-till systems allow maintenance of row position and should be amenable to controlled traffic. With equipment for eight 76-cm (30-inch) rows, for instance, the wheel track area would occupy only

TABLE X Compaction Effects on Corn Yields in Two Types of Soil in Central Illinois' Corn yields in kg/ha (bu/A) for nitrogen rates of: Soil type

Density level

Air voids 0-30 cm (0-12 inches) (%)

Drummer silty clay loam (typic haplaquoll)

Medium High

22 11

5210 (83) 4457 (71)b

7783 (124) 7030 (112)b

Thorp silt loam (argiaquic argiaboll)

Medium High

18 12

4017 (64) 3703 (59)

6654 (106) 6591 (105)

'From Bateman (1962). bSignificant difference from the medium density level.

44.8 kg/ha (40 Ib/A)

134.5 kg/ha (120 Ib/A)

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C. B. RICHEY ET AL.

about 17% of the total area, leaving 83% uncompacted except for implement action. The harvester should use the same wheel tracks and harvest eight 76-crn (30-inch) rows. Soybeans are considered to be more sensitive to compaction than corn, although there has been little research in this area. The current interest in deeper chiseling in the corn belt could be primarily due to farmer awareness of reduction in soybean yields due to compaction. The advent of 130 kW (175 hp) two-wheel drive tractors and combines weighing up to 13,500 kg (30,000 Ib) loaded provides capability of deeper soil compaction than previously experienced. Additional tire area does not greatly reduce deep compaction because the pyramid of support is principally affected at the surface. Gill (1971) states: Soil compaction is a national problem of significant consequence in that it cannot be economically assessed accurately at this time. Compact soil conditions can naturally decrease the yields of crops over vast areas of tilled land. Increase in machinery size and use increase the seriousness of this problem. . . . The maintenance of a low degree of soil compaction must be undertaken by the development of complete crop production systems. The recompacting actions of operations subsequent to initial soil loosening operations, in excess of any desirable firming operations, must be eliminated from the crop production system when economically justifiable in order to prevent a gradual degeneration of a desirable loose condition.

C. WEED CONTROL

Tillage can help control weeds by: 1. Burying weed seed and delaying perennial weeds. 2. Leaving a rough surface to discourage weed seed germination. 3. Providing enough loose soil to permit effective row cultivation. 4. Leaving a clean uniform surface for efficient herbicide action. 5. Incorporating herbicides when necessary. Plow tillage systems are the most effective in controlling weeds since they comply with items 1, 2, and 4 above. Several herbicide-resistant perennial weeds such as milkweed (Asclepias syriacu L.) which are kept under control by plowing, may become serious pests when plowing is omitted for several years (Williams and Wicks, 1976). Chisel systems do not bury weed seed as well as plow systems and leave more residue on the surface. Field experiments in Indiana (Bauman, 1976) found that corn plant residue covering 85% of the soil surface resulted in only 70% of atrazine spray reaching the soil surface for weed control. Not only was 30% of the herbicide intercepted by the residue but many areas under residue were untreated.

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In the dry spring of 1976, some farmers in Indiana and Illinois noted that corn following soybeans showed less trifluralin carry-over damage where plowed than where chiseled. Presumably chiseling did not dilute and bury the old surface soil as well as plowing and there was not enough rainfall to carry the herbicide down away from the young plants. Conventional disks cutting only 10 cm (4 inches) deep leave a fme seedbed which encourages weed germination and do not bury weed seeds. Herbicide rates must approach those for no-till to obtain good weed control. The till-plant system provides rough inverted middles which tend to discourage weed growth. Weed germination is high in the smooth row area but herbicides are effective. The row weeds can also be controlled by a rolling cultivator w h c h can work through the rough middles without clogging. The ridge system with residue in the furrow has allowed reasonably good weed control. Ridge reshaping in the fall provides some inversion of weed seed, a clear row area for herbicides and some loose soil for cultivation. The weed problem builds up year after year with continuous no-till coulter and rotary strip tillage systems. Herbicide-resistant weeds, such as milkweed (Asclepias syriaca L.), fall panicum (Panicurn diehotomiflorurn Michx.), and briars are naturally selected. A no-till coulter plot was plowed the fourth year in the Indiana experiments and then continued with no-till. During the fifth and sixth years it showed a noticeable reduction in weed population compared t o the continuous no-till plot. In Ohio experiments (Van Doren et al., 1976) “Most of the weed control problems occurred in the no-tillage plots of the corn-oats-meadow rotation. During the first six years, about 40% of all no-tillage plots had problems compared to 10% for plowed plots. Problems were cut in half during the remaining years, indicating improved, but not yet perfect ‘state of the art,’ especially for no tillage .” In Table VII, weed weights are shown for Illinois soybean plots in 1974 (Siemens and Oschwald, 1975). Plowed plots showed a great advantage over the other plots, although all plots were rated as having good weed control. The no-till system has a serious handicap in that mechanical cultivation is usually ineffective because of the firm soil. Thus total reliance must be placed on herbicides, whereas cultivation can “back stop” herbicide failure in the other systems. This problem should be minimized as more effective herbicides are developed,

D. FERTILIZER PLACEMENT

Obviously, nutrients must be available at locations in the soil accessible to plant roots. With the no-till coulter system, broadcast fertilizer tends to stay

164

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near the surface. Due to the firm soil and moisture available near the surface under the mulch, roots tend to feed closer to the surface than with conventional tillage, with consequent vulnerability to dry weather. In the Indiana experiments cited earlier, nitrogen deficiency was noted, both visually and by leaf analysis, in certain years prior to 1971 in strip-tillage plots on poorly drained soils. In 1971-1972, conventional, chisel, and coulter plots on poorly drained Runnymede loam were split three ways. One-third of each plot received NH3 d e e p placed, one-third received liquid N on the surface plus a knife through the soil with no N deep-placed, and one-third received surface N only. The same rate of N, 168 kg/ha (150 lb per acre) was used with each treatment. Special adaptations to apply NH3 in no-till coulter plots included a separate independently mounted coulter to cut through trash and sealing wings welded to each knife at two levels. Deep-placed N brought no-till yields up to conventional tillage for the 2 years of this study. Tillage effect of the NH3 knife also appeared to increase yields when N was surface applied on noncultivated no-till plots. There was some indication of improved yield for deep-placed NH3 in chisel plots, but stand variability also influenced yield. Maturity of no-till corn was not improved by N placement. Grain moisture at harvest was 2% higher in 1971 and 5% higher in 1972 for no-till versus conventional corn with all N placement treatments. In Illinois experiments comparing plow, chisel, disk and no-till treatment for corn (Siemens et d.,1971) soil tests for pH, P, and K were made at 0- to 8-, 8to 1.5, and 15- to 23-cm (0- to 3-, 3- to 6-, 6- to 9-inch) levels. Total nutrients did not vary significantly. The plowed plots were fairly uniform in the first two levels, dropping to 73% in the third level. The no-till plots had only about one-half the nutrients in the second level and about 40% in the third level of those in the first level. The other treatments were similar to the no-till. It was concluded that: “Moldboard-plowing and subsequent mixing of the soil to plow depth may be necessary for high corn yields in years when the surface soil is extremely dry and plants are obtaining the majority of their phosphorus and potassium from lower depths.” With conventional tillage, phorphorus and potassium can be broadcast and worked in by subsequent plowing or chiseling. Ridging after broadcasting tends to concentrate the fertilizer in the ridge. This would seem to be advantageous, especially for starting young plants, but has not been evaluated by experiments. Nitrification inhibitors make possible fall application of nitrogen fertilizers without overwinter leaching losses. Indiana experiments (Warren et al., 1975) with fall application of nitrogen found that the addition of the inhibitor nitrapyrin increased corn yields 24% for a rate of 85 kg/ha (76 lb/A) and 12% for a rate of 170 kg/ha (1 52 lb/A) and gave yields equal to the same amount of nitrogen applied in the spring.

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165

Application of nitrogen before fall tillage should result in less soil compaction than spring application as well as in reduction of spring work, although coulters and extra sealing wings may be needed to apply anhydrous ammonia through surface residue into firm soil.

E. MOISTURE CONSERVATION

Tillage affects soil moisture content by: 1. Facilitating drying by loosening the soil, thus enabling earlier planting. 2. Facilitating water intake by loosening the soil and leaving residue on the surface to reduce sealing and runoff. 3. Reducing evaporation by leaving residue on the surface. 4. Controlling weeds which use soil moisture. When tillage loosens soil but buries residue, the net effect is usually reduced soil moisture. This is evidenced by the advantage of no-till coulter planting over conventional tillage in sod-planted corn (Smith and Lillard, 1976; Blevins et aZ., 1971) and in double-cropping soybeans or grain sorghum after small grain, where moisture for germination is critical. Experiments in Indiana (Mannering er al., 1975) determined that residue cover after planting corn following corn was 0.6-1.8% of the total surface for conventional plow tillage, 4-14% for till-plant, 10-18% for disk, 6-29% for chisel, 38-89% for strip rotary, and 59-82% for no-till coulter. Runoff from artificial rainstorms of 10 cm (2.5 inches) per hour was greatest on plowed plots and least on chiseled plots with no-till plots in between. Water content in the top 19 cm (27 inches) of soil throughout the season was usually greatest for the no-till plots and least for the plowed plots. The results from these tests show limited tillage systems to be effective in moisture conservation for two principal reasons: (1) Those systems that leave the surface rough and porous or create ridges at right angles to major slopes effectively increase infiltration, thus reducing runoff. In addition, the rougher the surface the more tortuous the runoff path and the slower the velocity of runoff thus allowing more time for infiltration. The roughness factor disappears with time during the season-the rate being dependent upon the stability of the soil material. (2) Those systems that leave large portions of the soil surface covered with residues offer surface protection from raindrop detachment of soil particles and surface sealing which limits water intake. The residues also serve as barriers to runoff thereby decreasing its velocity, again allowing more time for infiltration. Systems that rely on surface residues for protection are likely to be more permanent than those dependent on soil roughness. Several systems such as the chisel and till plant contain the roughness factor plus some residue effect. . . . These results support previous work showing those tillage systems that leave a large portion of the soil surface covered with residue to be very effective in reducing

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evaporation losses, thus conserving soil moisture. This serves as a distinct advantage for crop production on droughty soils, while on poorly drained sites the greater soil moisture content under residue in the spring results in delayed planting because of wetness and reduces plant growth because of cooler soil temperatures. (Mannering el al., 1975)

As can be seen from Table 111 the clean-tilled plow systems have not yielded as well on the light-colored droughty Bedford soil in southern Indiana as have the other systems, all of which leave varying amounts of residue on the surface and do not loosen and dry out the soil as much. The yields of the strip tillage systems rank near the top rather than at the bottom as in northern Indiana. This is apparently because the early-season temperature depression is less serious and is outweighed by the later moisture conservation effects. F. SOIL EROSION PREVENTION

It has been well established that soil erosion tends to be inversely proportional to the percent of the soil surface covered by plant-residue mulch (Wischmeier, 1973). A mulch cover facilitates infiltration by protecting the soil surface from raindrop impact and consequent sealing over. It also reduces runoff velocity which both increases infiltration and reduces transport of soil particles. In Ohio studies (Harrold and Edwards, 1974) soil erosion was monitored from 1949 to 1969 on two comparable watersheds of about 1.5 ha (0.6A), each under a corn, wheat, meadow, meadow rotation. Soil loss averaged about 2250 kg/ha (2000 lb/A) per year on each throughout the period. Continuous corn was then grown for the next 4 years, one plot with conventional plow tillage and the other with no-till coulter planting. Soil loss averaged 5980 kg/ha (5340 lb/A) per year on the conventionally tilled plot and 64 kg/ha (58 lb/A) on the no-till plot. Experiments in Indiana (Meyer and Mannering, 1961) compared infiltration and soil loss on conventional plowed and disked plots with plowed and wheeltrack planted plots. Rainulator (artificial rainfall) tests totaling 13 cm (5.2 inches) at a 6.5-cm (2.6-inch) per hour rate shortly after planting gave a soil loss of 37.4 tonnes (t)/ha (16.7 tons [TI /A) for the conventional and 19.5 t/ha (8.7 T/A) for the reduced tillage plots. The tests were repeated when the corn was about 60 cm (24 inches) high and the conventional tilled and cultivated plots lost 23.3 t/ha (10.4 T/A), the reduced tillage uncultivated plots 33.4 t/ha (14.9 T/A). The Russell silt loam soil, 4.5 to 5.0% slope had a tendency to crust. This resulted in highest runoff and erosion on the uncultivated plots which had a heavy crust resulting from the previous rainulator tests. A third series of tests was made in September after harvest with broken over cornstalks in place. All plots lost less than 2.2 t/ha (1 T/A) with the uncultivated plots lowest because of a heavy grass cover.

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Experiments in southwestern Iowa (Moldenhauer et aZ., 1971) compared second-year corn on conventional plowed plots, till-planted plots, and ridge plots. In the Iowa system (Buchele et aL, 1955) the ridge is maintained by a summer cultivation, with planting on the ridge top preceded by shredding the stalks, which settle into the furrows. Rainulator tests were performed in June after the first cultivation on rows with slopes of 3.4%, 6.9%, and 9% in order to evaluate the various treatments for erosion control with off-contour rows. A total of 19 cm (7.5 inches) was applied by two storms on successive days with a maximum rate of 12.5 cm (5 inches) per hour. The means for all three slopes were: conventional, 14 cm (5.5 inches) runoff, 62.5 t/ha (27.9 T/A) soil loss; till-plant, 13 cm (5.2 inches) runoff, 48.6 t/ha (21.7 T/A) soil loss; and ridge, 15.5 cm (6.2 inches) runoff and 15.5 t/ha (6.9 T/A) soil loss. The conventional system had the most loose soil and the greatest soil loss. With the till-plant system the row area was lower than the rough middles, causing the water to run down the row. With the ridge system, the water ran down the furrow which had some mulch protection and little loose soil, resulting in lowest soil loss. Slope had little effect on amount of runoff but in the conventional and till-plant plots increasing slope from 3.4% to 9% approximately doubled the soil loss. Soil loss from the ridge plots was little affected by slope change. “As an off-contour conservation tillage method, the ridge system was much more effective than the other two systems because soil and water losses from the ridge system tended t o be independent of slope within the range tested.” Rainfall simulator tests on corn after corn plots in Illinois (Siemens and Oschwald, 1975), after planting showed soil losses ranging from 37.0 t/ha (16.5 T/A) for conventional tillage to 6.3 t/ha (2.8 T/A) for no-till coulter planting with chiseling averaging 11.O (4.9 T/A), as shown in Table V. The results for similar spring tests before spring tillage comparing plots previously in corn with those previously in soybeans gave the results shown in Table XI (Oschwald and Siemens, 1975). Runoff starts earliest on the treatment with the smoothest surfaces, such as those created by the fall plow, disk and chop-plant (no-till) treatments. The rough surface on the chisel treatments provided more resistance to runoff, so that more time was required to produce runoff. In addition, the total runoff quantity was smallest with the three chisel treatments. Soil loss was highest with the fall-plow treatment in all three storms. Crop residues on the surface and a rough stable surface reduced soil loss on the conservation tillage systems, compared to the fall-plow treatment. Soil loss was greater after soybeans than after corn with all tillage treatments. The larger quantity of residue on the surface following corn and the loose, easily eroded soil following soybeans helps account for the differences in soil loss between corn and soybeans. We concluded that conservation tillage systems which provide a protective cover of crop residues and a rough stable surface also protect the soil from excessive soil erosion.

TABLE XI Runoff and Soil Loss as Influenced by Water Applied, Kind of Fall Tillage, and Crop or Year. Catlin Silt Loam (typic argiudoll), 5% Slope‘

Time b (minutes)

Water appliedC cm (inches)

Fall moldboard plow

Corn (1974)

Soybeans (1975)

No fall tillage

Disk-chisel

Corn (1974)

Soybeans (1975)

Corn (1974)

Soybeans (1975)

2.3 (0.89) 4.6 (1.81) 7.1 (2.79)

3.2 (1.27) 6.0 (2.37) 8.8 (3.47)

0.42 (0.19) 0.76 (0.34) 1.12 (0.50) 2.3 (1.03)

1.38 (0.62) 2.59 (1.16) 3.86 (1.72) 7.9 (3.5)

Runoff, in cm (inches)

60 90 120

6.4 (2.5) 9.5 (3.75) 12.7 (5.0)

3.0 (1.19) 5.8 (2.29) 8.6 (3.37)

3.9 (1.53) 6.9 (2.71) 9.6 (3.78)

0.1 (0.04) 0.8 (0.31) 2.9 (1.13)

2.1 (0.83) 5.1 (2.00) 8.3 (3.25)

Soil loss, in tonnes/ha (T/A)

60 6.4 (2.5) 90 9.4 (3.75) 120 12.7 (5.0) Total soil loss:

4.0 8.6 12.7 25.3

(1.79) (3.84) (5.66) (11.3)

10.9 (4.87) 18.1 (8.06) 25.7 (11.4) 54.5 (24.3)

0.06 (0.03) 0.40 (0.18) 1.45 (0.65) 1.93 (0.86)

2.76 5.17 7.46 15.4

(1.23) (2.31) (3.33) (6.9)

‘Oschwald and Siemens (1975). bTime from start of water application. c . Smulated rainfall applied at intensity of 6 cm (2.5 inches) per hour after overwinter weathering but prior to any spring tillage.

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The impact is greater on soil loss than on runoff. The beneficial effects on soil erosion must be balanced against any detrimental effects on crop production. (Oschwald and Siemens, 1976)

G. INSECT AND DISEASE CONTROL Some insects and diseases overwinter in exposed crop residues, and burying residue should help reduce survival, although there is little experimental data available. Musick and Collins (1971) in Ohio found more Northern rootworm (Diabrotica longicornis) eggs with more surface residue but the survival rate was less than in plowed soil, possibly because of freezing. Root damage was lower for no-till corn. At one time, burial of cornstalks was considered extremely important for European corn borer control but control was not complete enough t o be effective. It has been noted that the population builds up with no-till but resistant hybrid varieties have helped greatly. Surface residue can add to disease possibilities if other factors are favorable because there may be earlier and more spore development. Many disease organisms are destroyed by being buried in soil. The corn leaf blights, Dipbdia ear rot, and Gibberella ear rot all tend to be more severe with unburied corn residue. Crop rotation is the best control for many insects and diseases because it interrupts buildup of populations. Corn insects and diseases d o not usually survive in soybeans, nor do soybean insects and diseases in corn.

V. Energy Requirements for Various Tillage-Planting Systems

A. OPERATIONS IN LOW-, MEDIUM-, AND HIGH-DRAFT SOILS Since tillage operations are notoriously variable in energy requirements, depending on the soil type and previous treatment, energy estimates are given for low-, medium-, and high-draft soils in Table XII. The estimates for conventional tillage and cultivating are based on Table 1 in Agricultural Machinery Management Data (1976). Draft estimates for low-, medium-, and high-draft soils were converted to drawbar kWh/ha (hphr per acre). PTO kWh/ha (hphr per acre) was then calculated using estimated tractive efficiencies between 50 and 70%, depending on soil type and condition. Fuel requirement was calculated using an estimate of 2.46 PTO kWH/liter (12.5 PTO hphr/gal) of diesel fuel.

TABLE XI1 Energy and Diesel Fuel Requirementsa for Various Tillage and Planting Operations

Soil draft classification

Low Operation

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Shredding stalks Moldboard plowing, 8” deep Chisel plowing, 8“ average depth Disking stalks Disking tilled ground Ridge (furrow mulch) Field cultivating tilled ground Planting, conventional Wheel-track planting Field cultivate-planter combination Till-planting Strip rotary till-planter combination No-till (fluted coulter) planting Rotary hoeing Row-crop cultivating, conventional Row-crop cultivating, till-planted

Medium

High

PTO kWh/ha (PTO hphr/A)

l/ha (gal/A)

PTOk Wh/ha (PTO hphr/A)

1/ha (gal/A)

PTOkWH/ha (PTO hphr/A)

(gal/A)

18.5 (10) 33.2 (18) 22.2 (12) 10.2 (5.5) 11.1 (6) 33.2 (18) 11.4 (6.2) 9.2 (5) 11.4 (6.2) 20.7 (11.2) 9.2 (5) 12.9 (7) 9.6 (5.2) 3.1 (2) 4.6 (2.5) 6.1 (3.3)

7.5 (0.8) 13.1 (1.4) 8.9 (0.95) 4.2 (0.45) 4.7 (0.5) 13.1 (1.4) 4.7 (0.5) 3.7 (0.4) 4.7 (0.5) 8.4 (0.9) 3.7 (0.4) 5.1 (0.55) 4.2 (0.45) 1.4 (0.15) 1.9 (0.2) 2.3 (0.25)

18.5 (10) 53.5 (29) 35.1 (19) 9.2 (5) 12.9 (7) 40.6 (22) 23.1 (12.5) 11.4 (6.2) 14.8 (8) 24.9 (13.5) 11.4 (6.2) 16.6 ( 9 ) 12.0 (6.5) 5.5 (3) 5.9 (3.2) 7.9 (4.3)

7.5 (0.8) 21.5 (2.3) 14.0 (1.5) 3.7 (0.4) 5.1 (0.55) 16.4 (1.75) 9.4 (1.0) 4.7 (0.5) 6.1 (0.65) 10.3 (1.1) 4.7 (0.5) 6.6 (0.7) 4.7 (0.5) 2.3 (0.25) 2.5 (0.27) 3.3 (0.35)

18.5 (10) 73.8 (40) 48.0 (26) 9.2 (5) 14.8 (8) 48.0 (26) 35.1 (19) 13.8 (7.5) 22.2 (12) 31.4 (17) 15.2 (8.2) 23.1 (12.5) 15.7 (8.5) 7.4 (4) 7.9 (4.3) 10.5 (5.7)

7.5 (0.8) 30.0 (3.2) 20.0 (2.1) 3.7 (0.4) 6.1 (0.65) 19.7 (2.1) 14.0 (1.5) 5.6 (0.6) 8.9 (0.95) 13.1 (1.4) 6.1 (0.65) 9.4 (1.0) 6.6 (0.7) 2.8 (0.3) 3.3 (.35) 4.2 (0.45)

~~~~

~

~

~~~

‘Tractor fuel consumption was taken as 2.46 kWh/l (12.5 PTO hphr per gallon).

l/ha

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171

The estimates for the various planting methods are based primarily on measurements made by the authors in 1970 (Richey et d.,1973). Engine power was measured by setting the diesel tractor throttle for the same no-load rpm before all tests and then measuring fuel consumption and time for a measured distance during the operation. The tractor was then calibrated with varying loads on an electric PTO (power take-off) dynamometer, using the same no-load rpm setting, to obtain a curve of PTO power versus fuel rate. This along with ground speed allowed PTO kWh/ha (PTO hphr per acre) t o be calculated.

B. VARIOUS TILLAGE-PLANTING SYSTEMS IN LOW-, MEDIUM-, AND HIGH-DRAFT SOILS

Estimates of energy and diesel fuel requirements for the high-energy, moderate-energy, and low-energy tillage-planting systems described previously are shown in Table XIII, Table XIV, and Table XV, respectively. The field operations are indicated by the identification number assigned in Table XI1 and total €TO kWh/ha (PTO hphr/A) and liters per hectare (gallons per acre) of diesel fuel are shown. Herbicides are listed with their energy requirements in diesel fuel equivalent, based on an average of 66.11 kWh/kg (25,800 kcal/lb) (Gunkel et al., 1976) of active ingredient with its carrier. The energy content of diesel fuel is taken as 11.35 kWh/liter (36,958 kcal/gal) (Wittmus and Lane, 1973) giving 5.82 liters diesel fuel equivalent for 1 kg (0.7 gal/lb) of active herbicidal material and its carrier. These are added to the figures for field operations to obtain the total cultural energy requirement in F'TO kWh/ha (PTO hphr/A) and liters/ha (gallons/A). Variations in operations can be readily evaluated by using the information in Table XII. Total cultural energy requirements in diesel fuel equivalent for each tillageplanting system and on low-, medium-, and high-draft soils are summarized in Table XVI for corn and in Table XVII for soybeans. Corn is considered to follow corn and soybeans are also considered to follow corn.

C. CORN TILLAGE SAVINGS

Moldboard plowing with wheel-track planting saves as much energy as no-till in low- and medium-draft soils, due to the elimination of secondary tillage and the use of banded herbicide instead of broadcast. It requires, however, much greater plowing capacity to keep ahead of planting, with consequent high fixed cost for the additional equipment investment.

TABLE XI11 Energy and Diesel Fuel Requirementsa for High-Energy Tillage-Planting Systems

Soil draft classification

w 4 2 .t

Low Herbicide active ingredient Tillageplanting system*

W h a (WA)

PTOkWhha (PTO hphr/A)

Medium l/ha (gal/A)

PTO kWh/ha (PTO hphr/A)

High l/ha (gal/A)

PTOkWh/ha (PTO hphr/A)

l/ha (gal/A)

(57.4)

42.8 (4.57)

134 (72.8)

54.6 (5.82)

89.0 (48.4)

7.9 (0.84) 13.1 (1.4) 63.8 (6.81) 36.4 (3.89)

121 (65.6)

10.5 (1.12) 13.1 (1.4) 78.2 (8.34) 49.1 (5.25)

~

Corn Moldboard plow. Operations 4, 2, 5, 5, 8, 15 Atrazine Lasso Total Moldboard plow, wheel track plant. Operations 4, 2,9,15, 15 Atrazine (band) Lasso Total

79.4 (43) 1.12, 1.35, 1.79, (1, 1.2, 1.6) 2.24 (2.0) 64.0 (34.7)

0.38, 0.45, 0.61 (0.34, 0.4, 0.54) 0.53 (0.4)

32.3 (3.44) 6.6 13.1 52.0 25.7

(0.7) (1.4) (5.54) (2.75)

2.2 (0.24) 4.4 (0.47) 32.3 (3.50)

106

2.6 (0.28) 4.4 (0.47) 43.4 (4.64)

3.6 (0.38) 4.4 (0.47) 57.1 (6.1)

Chisel plow. Operations 4 , 3 , 5, 5, 8, 15 Atrazine Lasso Total

r -1

Soybeans Moldboard plow. Operations 4 , 2 , 5 , 5 , 8, 14, 15, 15 Lorox Lasso Total Chisel plow. Operations 4 , 3 , 5 , 5 , 8 , 14, 15, 15 Lorox Lasso Total

72.8 (39.4) 1.4, 1.68, 1.79 (1.25, 1.5, 1.6) 2.24 (2.0)

95.1 (51.5)

8.2 (0.88) 13.1 (1.4) 50.9 (5.43) 87.7 (47.5)

35.7 (3.8)

81.1 (43.9)

5.0 (0.53) 13.1 (1.4) 53.8 (5.73) 33.0 (3.51)

0.84, 1.4, 1.68 (0.75, 1.25, 1.5) 2.24 (2.0)

0.84, 1.4, 1.68 (0.75, 1.25, 1.5) 2.24 (2.0)

29.6 (3.15)

5.0 (0.53) 13.1 (1.4) 51.1 (5.44)

38.9 (4.14)

119.5 (64.7)

10.5 (1.12) 13.1 (1.5) 72.2 (7.70)

9.8 (1.05) 13.1 (1.4) 61.8 (6.59) 117.2 (63.5)

48.0 (5.12)

105.5 (57.2)

8.2 (0.88) 13.1 (1.4) 69.3 (7.4) 43.3 (4.61) 8.2 (0.88) 13.1 (1.4) 64.6 (6.89)

48.6 (5.18)

149.6 (81.0)

60.8 (6.48)

134.8 (73.0)

9.8 (1.05) 13.1 (1.4) 83.7 (8.93) 54.8 (5.84) 9.8 (1.05) 13.1 (1.4) 77.7 (8.29)

‘Diesel fuel equivalent for herbicides based on 66.1 1 kWh/kg (25,800 kcal/lb) of active ingredient plus its carrier and with diesel fuel at 11.35 kWh/l (36,958 kcal/gal), giving 5.82 l/kg (0.7 gal/lb). bOperations are those identified in Table XII. Herbicide rates are current Purdue recommendations.

TABLE X N Energy and Diesel Fuel Requirements' for Moderate-Energy Tillage-Planting Systems

Soil draft classification Herbicide active ingredient Tillageplanting systemb

c. 4 P

Corn Disk. Operations 4 , 5 , 5 , 8, 15, 15 Atrazine Lasso Total Ridge (furrow mulch) Operations, 6, 15, 8, 15 Atrazine Lasso Total

k s F a Ob/A)

Low

ash

Medium

~

~

~

PTOkWh/ha (PTO hphr/A)

]/ha (gal/A)

PTOkWh/ha (PTO hphr/A)

l/ha (gal/A)

PTOkWh/ha (PTO hphr/A)

]/ha (gal/A)

50.8 (27.5)

20.7 (2.25)

58.3 (31.6)

24.1 (2.54)

68.5 (37.1)

27.8 (3.0)

51.7 (28)

8.8 (0.94) 14.8 (1.58) 44.3 (4.77) 21.0 (2.2)

63.9 (34.6)

10.5 (1.12) 14.8 (1.58) 49.4 (5.24) 26.4 (2.79)

77.7 (42.1)

10.5 (1.12) 14.8 (1.58) 53.1 (5.7) 31.6 (3.4)

1.5, 1.79, 1.79 (1.34, 1.6, 1.6) 2.52 (2.25)

1.5, 1.79, 1.79 (1.34, 1.6, 1.6) 2.52 (2.25)

10.5 (1.12) 14.8 (1.58) 51.7 (5.49)

8.8 (0.94) 14.8 (1.58) 44.6 (4.72)

10.5 (1.12) 14.8 (1.58) 56.9 (6.1)

Soy beans Disk. Operations 5, 5, 8, 14, 15, 15 Lorox

Lasso Total Ridge (furrow mulch) Operations 6, 15, 8, 15, 15 Lorox Lasso Total

44.3 (24)

18.0 (1.95)

56.3 (30.5)

6.5 (0.7) 14.8 (1.58) 39.3 (4.23) 22.9 (2.4)

1.12, 1.4, 1.68 (1.0, 1.25. 1.5.) 2.52 (2.25)

1.12, 1.4, 1.68 (1.0, 1.25, 1.5) 2.52 (2.25)

6.5 (0.7) 14.8 (1.58) 44.2 (4.68)

54.6 (29.6)

22.6 (2.39)

69.8 (37.8)

8.2 (0.88) 14.8 (1.58) 45.6 (4.85) 29.0 (3.06) 8.2 (0.88) 14.8 (1.58) 52.0 (5.52)

66.6 (36.1)

27.1 (2.9)

85.7 (46.4)

9.8 14.8 51.7 34.8

(1.05) (1.58) (5.53) (3.71)

9.8 (1.05) 14.8 (1.58) 59.4 (6.34)

'Diesel fuel equivalent for herbicides based on 66.11 kWh/kg (25,800 kcal/lb) of active ingredient plus its carrier and with diesel fuel at 11.35 kWh/l (36,958 kcal/gal), giving 5.82 l/kg (0.7 gal/lb). boperatiom are those identified in Table XII. Herbicide rates are current Purdue recommendations.

TABLE XV Energy and Diesel Fuel Requirementsa for Low-Energy Tillageplanting Systems

Soil draft classification Herbicide active ingredient Tillageplanting systemb

c. 4 VI

Corn Till-plant. Operations 1, 11, 16 Atrazine Lasso Total No-till Coulter Operations 1, 13 Atrazine

Lasso Paraquat Total Rotary-20 cm Strip Operation 12 Atrazine Lasso Paraquat Total

kg/ha (WA)

Low

High ~

PTO kWh/ha (PTO hphr/A)

l/ha (gal/A)

PTO kWh/ha (PTO hphr/A)

l/ha (gal/A)

PTOkWh/ha (PTO hphr/A)

l/ha (gal/A)

33.8 (18.3)

13.7 (1.45)

37.8 (20.5)

15.4 (1.65)

44.1 (23.9)

17.9 (1.0)

28.1 (15.2)

8.8 (0.94) 14.8 (1.58) 37.3 (3.97) 11.4 (1.25)

30.5 (16.5)

10.5 (1.12) 14.8 (1.58) 40.7 (4.35) 12.4 (1.3)

34.2 (18.5)

10.5 (1.12) 14.8 (1.58) 43.2 (4.60) 13.9 (1.5)

12.9 (7.0)

10.5 (1.12) 16.4 (1.75) 3.3 (0.35) 41.6 (4.47) 5.2 (0.55)

16.6 (9.0)

10.5 16.4 3.3 42.6 6.8

23.1 (12.5)

10.5 (1.12) 16.4 (1.75) 3.3 (0.35) 44.2 (4.72) 9.4 (1.0)

1.5, 1.79, 1.79 (1.34, 1.6, 1.6) 2.52 (2.25)

1.79 (1.6) 2.8 (2.5) 0.56 (0.5)

1.79 (1.6) 2.8 (2.5) 0.56 (0.5)

Medium ________

~

10.5 16.4 3.3 35.4

(1.12) (1.75) (0.35) (3.77)

(1.12) (1.75) (0.35) (4.52) (0.7)

10.5 (1.12) 16.4 (1.75) 3.3 (0.35) 37.0 (3.92)

10.5 (1.12) 16.4 (1.75) 3.3 (0.35) 39.6 (4.22)

(continued)

TABLE XV (continued) Soil draft classification Herbicide active ingredient Tillageplanting systemb

k g b (WA)

Soybeans No-till Coulter Operations 1, 13

Lorox Lasso Paraquat Total Rotary-20 cm Strip Operation 12 Lorox Lasso Paraquat Total

Low

High

PTOkWh/ha (PTO hphr/A)

l/ha (gal/A)

PTOkWh/ha (PTO hphr/A)

l/ha (gal/A)

PTOkWh/ha (PTO hphr/A)

l/ha (gal/A)

28.1 (15.2)

11.4 (1.25)

30.5 (16.5)

12.4 (1.3)

34.2 (18.5)

13.9 (1.5)

12.9 (7.0)

6.5 (0.7) 16.4 (1.75) 3.3 (0.35) 37.6 (4.05) 5.2 (0.55)

16.6 (9.0)

8.2 16.4 3.3 40.3 6.8

23.1 (12.5)

9.8 (1.05) 16.5 (1.75) 3.3 (0.35) 43.4 (4.65) 9.4 (1.0)

1.12, 1.4, 1.68 (1.0, 1.25, 1.5) 2.8 (2.5) 0.56 (0.5)

1.12, 1.4, 1.68 (1.0, 1.25, 1.5) 2.8 (2.5) 0.56 (0.5)

Medium

6.5 (0.7) 16.4 (1.75) 3.3 (0.35) 31.4 (3.35)

(0.88) (1.75) (0.35) (4.28) (0.7)

8.2 (0.88) 16.4 (1.75) 3.3 (0.35) 34.8 (3.68)

9.8 (1.05) 16.4 (1.75) 3.3 (0.35) 38.9 (4.15)

aDiesel fuel equivalent for herbicides based on 66.1 1 kWh/kg (25,800 kcal/lb) of active ingredient plus its carrier and with diesel fuel at 11.35 kWh/l (36,958 kcal/gal), giving 5.82 l/kg (0.7 gal/lb). bOperations are those identified in Table XII. Herbicide rates are current Purdue recommendations.

177

YIELDS AND REQUIREMENTS FOR CORN AND SOYBEANS TABLE XVI Total Energy for Corn Tillage, Planting, and Weed Control in Diesel Fuel Equivalent, l/ha (gal/A)

Soil draft classification Tillage-planting system

Low

Medium

High

Moldboard plow-conventional Moldboard plow-wheel track plant Chisel plow-conventional Disk-conventional Ridge (furrow-mulch) Till-plant No-till coulter Rotary strip

52 (5.54) 32.3 (3.46) 50.9 (5.43) 44.3 (4.77) 44.6 (4.72) 37.3 (3.97) 41.6 (4.47) 35.4 (3.77)

63.6 (6.79) 43 (4.6) 61.6 (6.57) 49 (5.2) 49.6 (5.27) 40.7 (4.35) 42.6 (4.52) 37.0 (3.92)

78.2 (8.34) 57.1 (6.1) 72.2 (7.70) 53.1 (5.7) 55.2 (5.92) 43.2 (4.6) 44.2 (4.72) 39.6 (4.22)

Till-planting requires less energy than no-till coulter planting, primarily due to lower herbicide requirements because of better inherent weed control than no-till. Erosion protection is not as good because of the bare depressed row area after planting. Chiseling with conventional secondary tillage saves little energy over plowing with conventional tillage, showing a maximum of 6 l/ha (0.65 gal/A) saving in heavy-draft soils. Disking and furrow-mulch ridging are quite similar in energy requirements. Savings compared to plowing are 7.6 l/ha (0.8 gal/A) for low-draft soils, 14.3 l/ha (1.55 gal/A) for medium-draft soils, and 24.3 l/ha (2.55 gal/A) for highdraft soils. Rotary strip tillage requires less energy than no-till because no stalk shredding is required. A rotary tiller is needed, however, in addition to a planter. No-till saves 10.4 I/ha (1.1 gal/A) over plowing and conventional tillage in low-draft soils, 21 l/ha (2.25 gal/A) in medium-draft soils, and 34.2 l/ha (3.65 TABLE XVII Total Energy for Soybean Tillage, Planting, and Weed Control in Diesel Fuel Equivalent, I/ha (gal/A) Soil draft classification Tillage-planting system Moldboard plow-conventional Chisel plow-conventional Disk-conventional Ridge (furrow-mulch) No-till coulter Rotary strip

Low

Medium

High

53.8 (5.73) 51.1 (5.44) 39.3 (4.23) 44.2 (4.68) 30.1 (3.25) 31.4 (3.35)

68.9 (7.36) 64.2 (6.85) 45.2 (4.81) 51.4 (5.46) 32.9 (3.48) 34.8 (3.68)

83.7 (8.93) 77.7 (8.29) 51.7 (5.53) 59.4 (6.34) 35.9 (3.85) 38.9 (4.15)

178

C. B. RICHEY ET AL.

gal/A) in high-draft soils. Unfortunately, no-till often has serious yield penalties in heavy soils although yields are often equal to conventional tillage on light soils, where energy savings are moderate.

D. SOYBEAN TILLAGE SAVINGS

Rotary hoeing and an extra cultivation are added for soybeans but herbicide energy is usually slightly less as compared to that for corn. The figures in Table XVI and XVII indicate that soybeans require about 4.7 l/ha (0.5 gal/A) more energy than corn with high-energy tillage, slightly less with moderate-energy tillage and about 9.4 l/ha (1 gal/A) less with low-energy tillage. No-till shows 23.4 l/ha (2.5 gal/A) saving over plowing in low draft soil, 36.5 l/ha (3.9 gal/A) saving in medium draft soil, and 48.2 l/ha (5.15 gal/A) saving in heavy-draft soil. No-till has had little acceptance in soybeans, however, because of the weed control problem, except for double-cropping after wheat where moisture conservation is paramount. Chiseling saves 4.7-1 1.2 l/ha (0.5-1.2 gal/A) compared with plowing while ridging or disking saves 7.0-18.2 l/ha (0.75-1.95 gal/A). Chiseling is quite popular for corn or soybeans following soybeans since the ground is loose and residue is not troublesome. VI. Projecting Energy Savings with Reduced Tillage

As is apparent from the comparisons in Tables XVI and XVII, the maximum possible energy saving is equivalent to less than 42 l/ha ( 5 gal/A). At present prices this is equivalent to less than 25 kg/ha (1 bu/A) of corn. This is not a compelling economic incentive, particularly in cases where there is a substantial possibility of a yield reduction with reduced tillage. If, however, the price of fuel rises substantially relative to the price of corn, or more important, if fuel availability is reduced, all means of saving fuel must be considered. It has been estimated that, of Indiana’s 2,428,000-ha (6,000,000-A) 1976 corn crop, approximately 971,000 ha (2,400,000 A) could have used lowenergy tillage without reduced grain yields if pests were controlled (Griffith er al., 1976). It has also been assumed that the remaining hectarage (acreage) could adapt to medium-energy tillage such as disking or ridging in place of plowing and chiseling. On the basis of these estimates, possible Indiana energy saving has been projected as shown in Table XVIII. The projected saving averages about 9.1 l/ha (1 gal/A) for corn. Similar savings would appear to be possible for soybeans except for the problem of weed control. New herbicides may eliminate the need for cultivating

TABLE XVIII Possible Energy Savings in Indiana by Reduced Tillage for Corn, 2,428,170 ha (6,000,000 A) in 1976 Hectares (acres) Soil and climate adapted to low-energy tillage Herbicide-resistant perennial weeds Now using low-energy tillage Possible change to low-energy tillage Possible savings, assuming 50% medium-draft and 50% lowdraft soils, in changing from plow and chisel tillage, @ 14 l/ha (1.5 gal/A) Remainder adapted to moderate-energy tillage Herbicide-resistant perennial weeds Now using medium-energy tillage Possible change from high-energy tillage to moderate-energy tillage Possible savings, assuming 50% high-draft and 50% medium-draft soils, in changing from plow and chisel tillage @ 16.4 l/ha (1.75 gal/A) Possible total annual energy saving

Energy savings ](gal)

971,270 (2,400,000) 72,845 (180,000) 194,255 (480,000) 704,170 (1,740,000) 9,858,380 (2,610,000) 1,456,900 (3,600,000) 291,380 (720,000) 291,380 (720,000) 874,140 (2,160,000) 14,335,900 (3,780,000) 24:194,280 (6,390,000)

180

C. B. RICHEY ET AL.

soybeans, allowing solid-seeding as well as reduced tillage. Estimates have not been made for soybeans, however, because of the fluid state of the technology. An evaluation has been made of the adaptability of Ohio soils to reduced tillage (Triplett et al., 1973). Soil series were placed in five tillage groups based on yield response to the no-till system and also erosion vulnerability. Crop land totaled 3,809,915 ha (9,414,300 A) of which 1,349,000 ha (3,333,400 A) or 35% is rated to yield, with no-till, as well as or better than with conventional tillage. Another 25% is rated to yield almost as well with no-till where drainage has been improved, leaving 40% of the state’s cropland on which yields would be reduced by no-till. An accurate projection of energy savings in the United States by reduced tillage would require a similar analysis for all major crop-producing areas.

VII. Conclusions

1. Tillage increases yields primarily by facilitating early planting-germination-growth, weed control, and moisture conservation. The proportion of residue left on the surface by tillage is a major factor. 2. Moderate-energy tillage systems can be substituted for high-energy systems on most soils without yield penalty if pests can be controlled. 3. Low-energy tillage systems appear to be well adapted to the rolling soils of the southern corn belt and the well-drained loams and coarser textured soils of the northern corn belt if pests are controlled. Lowenergy tillage has not been well adapted to poorly drained soils. 4. Surface residue is the most effective deterrent to soil erosion and should be utilized wherever it does not result in economic penalties. 5. Energy savings with low-energy tillage are less than would appear because of (a) the energy required to produce the extra herbicide and (b) lack of adaption to high-draft soils, where the most saving would occur. 6. Overall energy savings from shifting to moderate and low-energy tillageplanting systems where practical is estimated to average about 9.1 l/ha (1 gal/A) for the Indiana corn crop. 7. The reduction in spring field work and the resulting reduction in late-planting yield penalties with moderate- and low-energy tillage-planting systems is probably more attractive to the farmer than energy savings.

REFERENCES Agricultural Machinery Management Data. 1976. Agric. Eng. Yearb. ASAE D 230.2,

322-329.

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Baeumer, K., and Bakermans, W. A. P. 1973. Adv. Agron. 25, 77-123. Barber, S. A. (1965). Res. Prog. Report, Purdue Agr. Exp. Sta. 168. Bateman, H. P. (1963). Trans. Am. Soc. Agr. Eng. 6(1), 19-25. Bauman, T. T. 1976. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Blevins, R. L., Cook, D., Phillips. S . H., and Phillips, R. E. 1971. Agron. J. 63,593-596. Bone, S. W., Rask, N., Forster, D. L., and Schurle, B. W. 1976. Ohio Rep. 61(4), 6 0 4 3 . Buchele, W. F., Collins, E. V., and Lovely, W. G. 1955. J. Agric. Eng. Res. 36, 324-329, 331. Dumas, W. T., Trouse, A. C., Smith, L. A., Kummer, F. A,, and Gill, W. R. 1973. Trans. A m . Soc. Agnc. Eng. 16,872-875, 880. Free, G. R., and Bay, C. E. 1964. Cornell Univ., Agric. Exp. Stn., Farm Res. Reprint 301. Gill, W. R. 1971. In “Compaction of Agricultural Soils” (K. K. Barnes, ed.), pp. 431-458. Am. SOC.Agric. Eng., St. Joseph, Michigan. Griffith, D. R., Mannering, J. V., Galloway, H. M., Parsons, S. D., and Richey, C. B., 1973. Agron. J. 65, 321-376. Griffith, D. R., Mannering, J. V., and Richey, C. B. 1976. Proc. Energy Agric. Conf., Cent. Biol. Nut. Syst,, Washington Univ., St. Louis,Mo., p. 162. Gunkel, W. W., Price, D. R., Casler, G. L., Lucas, G. M., Murray, D. L., Sutter, S . 1976. Cornell Univ. Agric. Ext. Eng. Bull. 406. Harrold, L. L., and Edwards, W. M. 1974. Trans. Am. Soc. Agric. Eng. 17,414-416. Lane, D. E., and Wittmuss, H. D. 1961. Nebr., Univ., Agric. Home Econ., Ext. Sew., Ext. Circ. 61-714. Mannering, J. V., Griffith, D. R., and Richey, C. B. 1975. Am. Soc. Agric. Eng., St. Joseph, Mich. Pap. No. 75-2523. Meyer, L. D., and Mannering, J. V. 1961. J. Agric. Eng. Res. 42(2), 72-75, 86, 87. Moldenhauer, W. C., Lovely, W. G., Swanson, N. P., and Curranee, H. D. 1971. J. Soil Water Consem. 26(.5), 193-195. Musick, G. L., and Collins, D. L. 1971. Ohio Rep. 56(6), 88-91. Oschwald, W. R., and Siemens, J. C. 1975. In “World Soybean Research” (L. D. Hill, ed.), pp. 84-81. Interstate Printers & Publishers, Danville, Illinois. Oschwald, W. R., and Siemens, J. C. 1976. Agron. Facts, Univ. Ill. SM-30. Pendleton, J . W., and Egli, D. B. 1969. Agron. J. 61, 70, 71. Poynor, R. R. 1950.J. Agric. Eng. Res. 31(9), 509, 510. Richey, C. B., Parsons, S. D., Griffith, D. R., Galloway, H. M., and Mannering, J. V. 1973. Am. Soc. Agric. Eng., St. Joseph, Mich. Pap. No. 73-113. Siemens, J. C., and Oschwald, W. R. 1975. In “World Soybean Research” (L. D. Hill, ed.), pp. 63-73. Interstate Printers & Publishers, Danville, Illinois. Siemens, J. E., Walker, W. M., and Peak, R. T. 1971. Ill., Agric. Exp. Stn., IlZ. Res. 13(3), 6, 7. Smith, E. S., and Lillard, J . H. 1976. Trans. Am. Soc. Agric. Eng. 19(2), 262-265. Triplett, G. B., Van Doren, D. M., and Bone, S. W. 1973. Ohio, Agric. Exp. Sm., Res. Bull. 1068. USDA Agricultural Statistics. 1900. p. 759. U.S. Gov. Printing Office, Washington, D.C. USDA Agricultural Statistics. 1942. p. 54. U.S. Gov. Printing Office, Washington, D.C. Van Doren, D. M., and Triplett, G. B. (1973). SoiESci. Am. Proc. 37, 766-769. Van Doren, D. M., Triplett, G . B., and Henry, J. E. 1976. Soil S c i Am. J. 40(1), 100-105. Warren, €I. L., Hubcr, D. M., Nelson, D. W., and Mann, 0. W. 1975. Agron, J. 67,655-660. Willard, C. J., Taylor, G. S., and Johnson, W. H. 1956. Ohio, Agric. Exp. Stn., Res. Circ. 30. Williams, J. L., Jr., and Wicks, G. A. 1976. Proc. Symp. Crop Residue Manage. Syst. Am. SOC.Agron. Spec. Publ.

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Wischmeier, W. H. 1973. Conserv. Tillage: Proc. Natl. ConJ pp. 133-141. Soil Conserv. SOC. Am., Ankeny, Iowa. Wittmuss, H. D., and Lane, D. E. 1973. Am. SOC. Agric. Eng., St. Joseph, Mich. Pap. No. 73-1 12. Wittmuss, H. D., Lane, D. E., and Somerhalder, B. R. 1971. Trans. Am. SOC.Agric. Eng. 14(1), 60-63,68.

EFFECTS OF THE ENVIRONMENT ON THE GROWTH OF ALFALFA K. R . Christian Division of Plant Industry. Commonwealth Scientific and Industrial Organization. Canberra. Australia

I . Introduction

.................................................. ........................ ShootGrowth ................................................. A. Mathematical Description ...................................... B . Internode Number ........................................... C. Leaf:Stem Ontogeny ......................................... D . Leaf Growth and Development .................................. RootGrowth .................................................. Environmental Factors and Vegetative Growth ........................ A. Light ..................................................... B. Temperature ................................................ C. Water ..................................................... D. Minerals ................................................... Phases in Development ........................................... A. Bud and Shoot Initiation ...................................... B . Root Carbohydrate Storage .................................... C. Regrowth Characteristics ...................................... D. Flower and Seed Formation .................................... E. Seedingandtheseed ......................................... Plant Associations .............................................. A . 1ntraspecific:Plant Density ..................................... B . Interspecific Competition ...................................... Genetic Adaptation to Environment ................................ References ....................................................

I1. Genetic Variation in Response to Environment

111.

IV . V.

VI .

VII . VIII

.

.

I

183 185 186 186 187 188 188 189 191 191 195 199 204 209 209 210 211 212 213 214 214 215 217 219

Introduction

The importance of alfalfa in world agriculture needs no further assertion than a reference to its venerable reputation since antiquity and to its geographical distribution . The future potential and limitations of this crop are t o be seen in the volume of scientific papers emanating from every major region in which its cultivation has been considered practicable . The agronomy of alfalfa in all its aspects has recently been described. both in the United States (Hanson. 1972) 183

184

K. R. CHRISTIAN

and in Australasia (Langer, 1967). A certain amount of repetition of the material contained in those extensive compilations will be unavoidable in this review, which is concerned with a more limited appraisal, from a nonspecialist perspective, of the complex of factors contributing to the variability in growth under different conditions. Plant growth might be described as a genetically planned construction attuned to the environment, if such a definition did not fail to convey fully the truism that the plant has no existence apart from the environment. When one speaks of the effect of environment, it is always a departure of some sort from some other environment that is implied. This leads naturally to the proposal of a standard or reference environment, which immediately raises problems in specification. The notion of an optimal environment is open to criticism, since the requirements for maximum dry matter yield are not necessarily those which produce material of best quality or which promote highest seed yields or greatest persistence. Furthermore, the ideal environment for any one of these objectives might well involve inordinate quantities of light and nutrients, C 0 2 enrichment, and so on. Nevertheless, any study of growth requires, tacitly or explicitly, a comparison with a “normal” behavioral pattern for that genotype under circumstances where development is not unreasonably hampered by any single external parameter. It is usually not practicable to specify the environment other than in general terms, such as a set of instrument readings at a given height above the crop, which may give little impression of the changes taking place beneath. The plant inhabits the two vastly differing media of atmosphere and soil, each of which influences the other. In partaking of the tetrad of earth, air, fire in the form of radiation, and water, the plant contributes to its own microenvironment at each level of the vertical profde, and thereby to the environment as a whole. Fredricksen (1938) described marked differences in runoff, soil moisture, soil structure, air temperature, humidity, and wind movement with an alfalfa field as compared with prairie bunch grass vegetation, and similar observations have been made since. The influence of the environment extends to the response of impinging organisms, including neighboring plants, pathogens, pollinators, symbiotic microflora, and grazing animals. Disease infestation is often a secondary effect, resulting from environmentally induced hazards such as waterlogging, frost damage, high temperatures, and nutrient deficiencies, but its importance may ultimately be much greater. Alfalfa plants grown in a pathogen-free environment can be subjected to severe clipping treatment without displaying plant mortality or root necrosis (Hamlen et al., 1972). Willis et al. (1969) showed that fungicide spraying could reduce leaf drop and increase hay yields by 18% over one growing season. Management practices such as tillage, irrigation, and fertilizer application, and variations in harvesting and grazing schedules, can also modify the environment

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

185

in many ways. In fact, it is difficult to think of aspects of growth which are not subject to environmental effects, except perhaps the genetic code itself; and even then, the interaction between genotype and environment is an important consideration.

11.

Genetic Variation in Response to Environment

The diversity and plasticity of alfalfa is illustrated by the fact that the cultivated forms cover the entire range between the extreme types of the Medicago sativa-falcata-glutinosa complex from which they were originally derived. The scope for further development of genetic material is seemingly unlimited. The strains containing Medicago falcata genes are generally characterized as winter-dormant and cold-hardy. Growth habit is somewhat prostrate, spreading, branching, and rosettelike at short day lengths, but erect and nonbranching like that of M. sativa at long day lengths. Other features of M. falcata genotypes include the tendency to form adventitious shoots on root segments (Smith, 1950), high shoot:root ratios (Heinrichs and Nielsen, 1966), and slow recovery after cutting (Tysdal and Kiesselbach, 1939). They are also thought to be more persistent, and more resistant to disease, waterlogging, and winter injury. Although it is tempting to think in terms of a “Medicago falcata syndrome,” evidence suggests that the association of these attributes is likely to be coincidental rather than due to genetic linkage. Cold-hardiness is usually regarded as an integral component of these genotypes, yet Greenham (1966) pointed out that different physiological factors are required at each critical stage, and that different genes may be responsible. It is difficult to see how these factors could not have developed contemporaneously during adaptation, but it is important to recognize that selection for only one of them may fail to achieve its overall objective. Likewise, the correlation between cold injury and growth at low temperatures has been interpreted as showing independent responses to natural selection (Daday, 1964). Winter dormancy appears to result from a qualitative difference in the form of a threshold for winter activity, rather than the expression of one extreme of a distribution of growth rates (Morley et al., 1957). Both erect, vigorous types and prostrate, slow-growing types are to be found in Mediterranean lines (Leach, 1970b). Persistence under frequent cutting may be attributable to a form of growth with broad crowns and shoots emerging close to the ground, rather than t o the M.falcara genes (Leach, 1971b; Cameron, 1974). The variability within populations illustrates the difficulty of classifying material according to genotype (Heinrichs e t al., 1969). On the other hand, classifications based on agronomic characters may be useful for making comparisons within local regions (Yamada and Suzuki, 1974), but extrapolation to other

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environments can be quite misleading. Zaleski (1954) classified as late-flowering several varieties which are regarded in the countries to which they are adapted as early-flowering, It is almost certainly because they were winter-active, and sustained such injury during more inclement conditions than those to which they were accustomed, that growth during spring was delayed because of their weakened condition. Song and Walton (1974) have suggested that breeding for late autumn growth, without the accompanying development of physiological adaptation to winter survival, can result in reduced plant vigor in the following spring. Furthermore, it is often impossible to tell how or even whether a particular phenotypic character contributes to survival. In the extreme climate of eastern Anatolia, for instance, wild forms of Medicago sativa with spreading, prostrate stems are found in the same area as forms which are perfectly erect (ChristiansenWeniger and Tarman, 1939). Citing the example of M. asiatica, which is highly resistant to leaf loss in Afghanistan, but undergoes severe leaf shedding when grown in Europe, Bennett (1 970) warns that “Except when employed for characteristics with a very high heritability (which can only be determined by prior genetic studies), or when conducted in an environment closely resembling that in which the collected material is to be utilized without further genetic manipulation, phenotypic selection is an unreliable basis for sampling.” Genetic+nvironment interactions represent possible sources of variation of uncertain magnitude to be kept in mind when discrepancies between reports are encountered. It is therefore remarkable that winter-dormant and winter-active types have been found to be not different in photosynthetic rates at different temperatures (Pearson and Hunt, 1972a) or in water-use efficiency (McElgunn and Heinrichs, 1975).

Ill. Shoot Growth

A. MATHEMATICAL DESCRIPTION

The following brief formal description indicates how one may proceed from measurements of differential growth to the quantitative assessment of total yield. The shoot is regarded as consisting of internodes, i in number, each of which contains a segment of stem of length 1 and cross-sectional area A , and two oppositely placed trifoliate leaves with thickness t and surface area S (one side only). The total aerial volume V of a plant with n shoots is then given by V=

5;

(IA t rS)

If V is made up of a proportion D of plant dry matter with density ~ ~ ( 1 . 8

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glml), according to Hundtoft and Wu (1970), W of water with density p w and void space A , the fresh weight Y is specified in appropriate units by where The formulas indicate the minimum requirements for the full interpretation of data, while the true picture will often be considerably more complex. No allowance is made for the incidence of branching, for example; nevertheless, a high correlation is likely to exist between leaf and internode numbers per stem (Liang and Riedl, 1964). Another problem is the high mortality of shoots during growth (Christian er al., 1970), representing considerable wastage of dry matter accumulation. Petioles, stipules, stem tips, flower buds, and other reproductive structures are reported to comprise a fairly constant 11% of total herbage dry matter from the bud stage onward (Fick and Holthausen, 1975). B. INTERNODE NUMBER

While the number of internodes which a plant produces may be characteristic in a given environment, the position at which flowering first occurs differs strikingly among phenotypes (Jones, 1950). Medler et al. (1955) concluded from a study of nine winter-hardy clones that flower positions at the 14th or 15th node were associated with long day length, while at short day lengths, flowers first appeared at about the 10th node. Using creeping-rooted clones under controlled conditions, Carlson (1965) found a similar relationship and noted that under long photoperiods plants continued to produce nodes even after starting to flower. In contrast, Dobrenz et al. (1965) found a high inverse correlation between minimum temperature and the number of nodes to the first raceme in a nonhardy variety “Moapa,” and to the time from cutting to floral initiation. However, Field and Hunt (1974) reported that the number of leaves and the node of first flower were unaffected by temperature at a photoperiod of 16.5 hours. It is difficult to reconcile this evidence except by postulating different responses of winter-dormant and winter-active varieties to day length and temperature. The contrasting experimental conditions may also have had some influence. Internode number is reduced by water restriction (Perry and Larson, 1974), and early transition from the vegetative to the reproductive stage may well be a response to stress, whatever its nature. The possible consequences on production of high temperatures close to dry and almost bare soil following cutting in summer warrant closer investigation.

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K. R. CHRISTIAN C. LEAF:STEM ONTOGENY

Lengths of internodes, and the ratios between them, are varietal characteristics in a given environment (Sheridan and McKee, 1968). In general, the length increases for the first few internodes, then steadily decreases toward the plant apex, while similar trends are apparent in leaf size. Leaf weight increases almost linearly with shoot height, with fairly constant chemical composition and uniformly high digestibility (Smith, 1970a; Christian et al., 1970). Because the stem has the function of a support, however, its cross-sectional area increases at the base, and the weight of stem dry matter increases almost as the square of the height (Christian er al., 1970). The top segments of shoots are of similar composition and of high digestibility, irrespective of shoot height, but make up only a small proportion of the total dry weight. The major weight gains during maturation are in the heavily lignified tissues of the lower stems, and hence nutritive quality falls off at an increasing rate as growth continues. The best time to harvest material of high digestibility is before severe leaf drop occurs and while the stems can still be readily cut at the base with a pair of scissors. Protein content is highly related directly to digestibility and inversely to lignin and cellulose contents. Weight increase of shoots is approximately linear over most of the period of vegetative growth (Pearson and Hunt, 1972d), although there appears to be an increase in the rate of stem thickening and elongation around the bud stage (Dent, 19.55; Nishikawa, 1966), with little increase in height or yield after the full bloom stage (Raguse and Smith, 1966). Large leaves are often associated with thick stems (Zaleski, 1954), and therefore probably with long internodes. Plants of Medicago fulcata type are usually regarded as leafier than M. sutiva types, but this may be merely due to an initially slower rate of stem development after cutting. Taken at the same stage of growth, different varieties have been found to have similar leaf stem ratios (Davies, 1960) and similar leaf and stem protein levels (Dobrenz er al., 1969). As maturity approaches, growth rates of M. falcata types become more rapid than those of M. sativa types, so that comparative yields become dependent on harvest date (Sprague and Fuelleman, 1941; Tysdal and Kiesselbach, 1939).

D. LEAF GROWTH AND DEVELOPMENT

Cell division is most active during the early stages of leaf expansion, and is inversely related to the rate of cell enlargement (Koehler, 1973). Eventually, at a particular leaf length, cell division ceases, and mean cell size then increases in proportion to leaf length. Leaf growth may therefore be modified in different ways at different stages of development, depending on whichever process is

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predominant. Area, weight, and photosynthetic activity all increase at comparable rates in developing leaves when expressed as proportions of the values at full expansion (Wolf and Blaser, 1971b). The rate of development, or number of days to full expansion, is related, naturally enough, to the rate of leaf and internode appearance (Wolf and Blaser, 1971b; Field and Hunt, 1974). Turrell (1942) classified alfalfa leaves as primary, secondary, and so on, according to the origin of the shoots that bore them (main stem, axillary bud from primary leaf, etc.). Leaves of higher order formed increasingly at the lower nodes as the plant aged; they tended to become successively less in area and thickness, with thinner epidermal, palisade, and spongy mesophyll layers. The earlier and larger leaves had high interna1:external surface ratios and high intercellular volume; although stomate density was lower than in smaller leaves, pore size was greater. These factors were hghly correlated with susceptibility to damage from SOz, indicating that gas exchange rates were proportionately higher in larger leaves. Evidently the smaller and later leaves are restricted at the cell enlargement stage, and are unlikely to become as photosynthetically effective. Considerable interest has been shown over the last decade in the measurement of specific leaf weight (SLW) as a possible index of photosynthetic activity. Designating leaf dry weight as YD, SLW may be expressed in terms of the formula given earlier as

SL W = Y D / S= t p D D Clearly, SLW may vary according to leaf thickness, dry matter content, or void space, or any combination of these. In Lolium varieties, SLW may be higher in thin leaves with small mesophyll cells than in thick leaves with large cells (Wilson and Cooper, 1969). Under and conditions, the desert shrub Enceliu furinosu has leaves of very high SLW, with compact mesophyll cells and very little intercellular space (Cunningham and Strain, 1969). In alfalfa, Delaney and Dobrenz (1974) found that SLW in different genotypes was related directly to the thickness of the leaf and of the palisade tissue, and inversely to leaf area. However, Barnes et al. (1969) concluded that SLW and leaf area were under separate genetic control, with all possible combinations being encountered.

IV. Root Growth

In the young seedling, root growth starts more slowly than shoot growth, and is mainly confined to the tap root, with little lateral development. Within 2-4 months, however, the shoot:root ratio declines from about 2.0 to 1.O (Gist and Mott, 1958; Matches et al., 1962). Thereafter, the ratio is likely to be governed

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to an increasing extent by environmental factors. After shoot maturation, tap root diameter and total weight continue to increase steadily (Crowder et al.., 1960; Nishikawa, 1965). Where the effective depth is 6 feet or less, maximum penetration depth may be attained within 1 year, with subsequent growth devoted to increasing the number and thickness of taproots and laterals (Upchurch and Loworn, 195 1). Thick lateral roots develop sporadically and secondary thickening extends down the whole length of the tap root (Tanaka, 1971a,b). Severing the tap root some inches below the soil surface results in increased lateral root development, with little effect on other growth (Klebesadel, 1964). Large varietal differences in the number of branch roots and in the proportion of primary roots showing branching have been observed (Smith, 1951). Little is known of the seasonal pattern of root growth. Jones (1943) distinguished the permanent or cambial roots, consisting almost entirely of secondary growth and providing transport and storage, from the transient roots, which are primary in structure and which undergo growth in spring and autumn and decay during summer. The typical pattern of root growth for many species was described by Loomis and Ewan (1936) as geotropic movement down to dry soil, followed by lateral branching in the moist layer above, without hydrotropic response. The process may be briefly described as an osmoregulated force extended by the plant (Creacen and Oh, 1972) against the mechanical resistance of the soil, which is primarily a function of bulk density and water content (Taylor and Gardner, 1963). A similar behavioral response apparently ensues when a sudden transition occurs to any inhospitable region, such as one of mineral deficiency or toxicity, or a hardpan layer. Where the restriction is less severe, growth is restricted to the tap root, and there is little or no lateral formation. The tap root always seems to develop first, even in compact soils, where branch roots eventually become more important (Carlson, 1925). On eroded slopes, the tap root may disintegrate, its functions being taken over by lateral roots (Lapinskiene, 1966). In deep sandy soils, there is little mechanical impediment, but the water-holding capacity is often low, encouraging tap root extension (Lamba et al., 1949; McCleUand, 1969). On fine textured soils, growth is often restricted by compaction, which may hamper growth by preventing the entry of root hairs or by lack of aeration (Scott and Erickson, 1964), and may be aggravated by animal traffic (Tanner and Mamaril, 1959). Increase in bulk density leads to lower plant yields (Cifford and Jensen, 1967), even when root growth is not visibly restricted (Peterson, 1971). Hence plants grown on clay soils are usually small (McClelland, 1969), with slower growth rates than on lighter soils (Dent, 1955), although the variety “Hairy Peruvian” is reported to be an exception to this rule (Rogers, 1963). Compaction generally increases with depth, which contributes to the

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

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frequent observation that most of the root system is contained within the first foot or so of soil (Lamba et aL, 1949; Upchurch, 1951; Bennett and Doss, 1960). Root penetration and water storage may be improved by mixing high density subsoil with topsoil (Cary e f al., 1967). In heavily compacted subsoils, tap roots follow cracks and cleavage planes (Fehrenbacher e t a l , 1965), often becoming thinner and crooked, but resuming normal growth further down (Scott and Erickson, 1964; Safta and Balan, 1971). In moist swelling subsoils, roots may follow earthworm burrows and gaps left by former decayed roots and evidently combine removal of water with root extension down the crevices left by the cracking of the soil mass (Fredricksen, 1938). The intervening blocks of soil are often bypassed, and the few lateral roots are confined to the more friable regions (Paltridge, 1955). Root growth is not limited to the vertical plane. Paltridge (1955) found that when roots were unable to penetrate a L'self-sealing'' layer, they spread laterally, up to 16 feet or until the roots of neighboring plants were reached. He affirmed that alfalfa is not necessarily or genetically a deep-rooting plant, but that its roots will grow in any plane where water is available.

V.

Environmental Factors and Vegetative Growth

A. LIGHT

I . DayLength Low growth rate under short day lengths is one of the most distinctive features of winter-dormant varieties. In particular, internode elongation is greatly reduced (Carlson, 1965). Much branching takes place, presumably as a result of loss of apical dominance, since at long day lengths, adventitious stem production is inhibited (Carlson, 1965). Since even normally developed stems in these varieties are often thin, the combination of reduced supporting ability and the change in load distribution probably contributes to the typically decumbent growth habit. The effects of day length and temperature overlap, and the interaction is likely to produce different responses in different varieties, but because of the great variability between individual plants, few detailed comparisons have been carried out. At temperatures of 15"/10°C (the figures written in this form will be used to indicate day and night temperatures, respectively) and 8-hour photoperiod, Sato (1971) found that leaves of Du Puits (an intermediate type) were thicker, but much shorter and narrower, than at higher temperatures or longer day lengths. As the temperature is raised, the growth inhibition by day length disappears. The data of Schonhorst et al. (1957) show that at 15.5"C the shoot

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height of winter-dormant strains does not increase when day length is extended from 8 to 12 hours, whereas in winter-active varieties at low temperatures and in all varieties at 26.7"C, shoot height is almost proportional to photoperiod. However, the results of Iversen and Meijer (1967) suggest first that weight responses may be much greater than responses in height, and second that the complete picture may be quite a complex one. Leaf development may be more rapid at long day lengths (Wolf and Blaser, 1971b) but leaf size is apparently not affected, and since stem diameter as well as length is increased (Sato, 1974), 1eaf:stem ratios are accordingly reduced (Coffmdaffer and Burger, 1958; Sato, 1971). Growth rates are reported to be highest under continuous light (Guy et uZ., 1971). Light interruption of the dark period was found to increase plant height during cooler, shorter days, with winter-active and intermediate types producing more erect and fewer semierect stems (Massengale et UZ., 1971). However, yields were in general higher with natural daylight only, particularly during the summer months. Reports on the effect of day length on root growth differ, suggesting that stage of growth or other interactions may be involved. Coffindaffer and Burger (1958) and Carlson (1965) observed no appreciable effect on root weights, and Seth and Dexter (1958) showed that there was little effect on tap root lengths of either hardy or nonhardy varieties. Hanson (1967) recorded increases in dry matter yields of all plant parts at long day lengths, although the proportion of roots was less. Sat0 (1971) found that root:top ratios tended to be highest at a 12-hour photoperiod. On the other hand, long day lengths have been observed to accelerate thickening of tap roots and growth of the root system as a whole while short days retarded root growth (Ueno and Tsuchiya, 1968). 2. Light Composition

Winter-active varieties increase in stem length much more rapidly than winterdormant varieties under mixtures of blue and red lights or green and red lights, particularly at low temperature (Nittler and Gibbs, 1959). Inhibition of hypocotyl elongation was also greatest in winter-dormant varieties. The diversity of reactions to selected colors found by these workers may indicate one reason for conflicting results between different laboratories and between the laboratory and the field. A complete, or at least a balanced, spectrum is required for maximum growth, particularly root growth (Heinrichs, 1973). The proportion of far red (740-mm) light transmitted by the alfalfa canopy is much higher than that of red (640-mm) light (Robertson, 1966); this would favor photomorphogenic reversal at the plant bases. It may also be significant that the proportion of far red radiation in natural sky light increases at twilight, particularly when the sky is clear; the effect will assume greater importance at

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high latitudes (Robertson, 1966). In Phaseolus, internode elongation is a function of phytochrome in the far red absorbing form at the start of night, and is proportional to the number of hours of darkness. The process differs from that due to long days and becomes less important at higher internode number (Vince-Prue, 1975). It seems possible that the initiation of new growth from the plant base could be inhibited by the canopy above through selective radiation effects as well as through apical dominance. Light intensity alone is insufficient to explain the effect, since bud elongation takes place in defoliated plants even in darkness and in plants where the crowns become exposed to direct sunlight because of lodging. There seems to be no evidence to suggest that changes in spectral composition during cloudy or overcast days are large enough to modify plant growth, but the possibility should perhaps not be overlooked. 3. Light Intensity Reduced light intensity removes growth inhibition by light, and the immediate result is rapid internode elongation (Pritchett and Nelson, 1951). The formation of the layer of woody tissue inside the cambium is reduced, so that the stems are much thinner than in normal plants and have the appearance of very young, succulent internodes. The effect is transient, coming to a halt more quickly at low light intensities, so that greatest stem height is likely to be found at intermediate levels (Pritchett and Nelson, 1951). Relative growth rates are increasingly reduced at light intensities below 50-75% of full daylight, and follow the general trend of the hyperbolic lightphotosynthesis curve. SLW increases with light intensity (Cooper and Qualls, 1967). Mean leaf area is diminished to a much lesser extent (McKee, 1962), so that the net effect may be that the leaf weight:total plant weight ratio remains fairly constant (Cooper, 1966, 1967). Stem growth is proportionately increased at the expense of root growth, particularly in the early stages of seedling growth (Cooper, 1966, 1967; Pritchett and Nelson, 1951). Nodulation is even more severely affected, presumably because of inadequate carbohydrate supply (Pritchett and Nelson, 1951; McKee, 1962). As shoot length increases, shading of lower leaves becomes an increasing limitation to production. Thomas and Hill (1949) found that net assimilation in plants 48 inches tall was only twice as great as in plants 6 to 8 inches tall, even though the leaf weight was 3.6 times as great. King and Evans (1967) observed that net photosynthesis in single plants approximately doubled as the leaf area index increased from 2 to 10. It should be noted, however, that the light response curves show that shaded leaves are photosynthetically just as efficient as younger, sunlit leaves at the light levels at which they are operating and,

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because of their low respiration rates (King and Evans, 1967), they are evidently not parasitic (Wolf and Blaser, 1971a). Experiments have shown that the longevity of leaves can be increased by thinning the stand (Wolf and Blaser, 1972; Pearce et al., 1968). However, removal of a large proportion of the photosynthetic area will almost certainly divert nutrients and growth factors to the remainder. Lower leaves continue to senesce even in full sunlight, but rejuvenation occurs within a few days if the upper part of the shoot is removed (Hodgkinson et al., 1972). The importance of apical dominance was further demonstrated by the observation that high rates of photosynthesis were maintained for a longer time when new shoot buds were removed as they appeared. Net photosynthesis per unit leaf dry weight was found by Pearce and Lee (1969) to be reasonably constant, whether differences in SLW were due to differences in environment or in genotype. The experiments of Delaney and Dobrenz (1974) also show that photosynthesis per unit leaf weight is not related to SLW. In the field, however, the decline in photosynthesis with the degree of shading experienced in dense canopies was much greater than that in SLW (Wolf and Blaser, 1971a). Pearce and Lee (1969) also observed that although photosynthesis was more constant on a leaf dry weight basis than on an area basis, other factors involving senescence resulted in different relationships in the field from those in the growth chamber. Changes in light intensity have been shown to result in very substantial adaptation within 2 weeks, both in photosynthesis and in SLW, with the result that photosynthesis per unit weight was much the same, irrespective of previous light treatment (Pearce and Lee, 1969). Similar trends were observed in canopies thinned to one stem per plant (Wolf and Blaser, 1972) although the response in photosynthetic rate was less at high light and greater at low light than the response in SLW.

4. m e Diurnal Cycle Increases in starch and sugar fractions of alfalfa herbage during daylight have been reported by a number of workers. The variability in the results may be partly attributed to analytical methods as well as to stage of plant growth and to environmental effects. Lechtenberg et al. (1971) found that the starch content of leaves increased by 10%during the day while that of stems showed almost no change, accounting for most of the concurrent increase in 1eaf:stem ratio from 1.1 to 1.5. Chatterton et al. (1972) observed that the rise in total nonstructural carbohydrates in daylight hours would account for 70% of the overall change in SLW. Concentrations fell during the first few hours of sunlight and increasei thereafter, with rapid responses to changes in light intensity.

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

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Chatterton (1973) also found that SLW and net carbon exchange showed closely related inverse trends during the day, and suggested that the buildup of assimilates could have been inhibiting photosynthesis. However, the data could equally be used to suggest that fluctuations in photosynthesis due to other causes would bring about corresponding changes in SLW. Pearson and Hunt (1972a) reported a slight decline in net carbon exchange in alfalfa after several hours of illumination under controlled conditions, but it occurred earlier at higher temperatures, when starch would be less likely to accumulate. Although feedback mechanisms have been suggested as possible limitations to photosynthesis on a number of occasions, the changes in concentrations have not been large enough to be particularly convincing. The reverse trend-the loss of carbohydrate and dry matter from the shoots during the hours of darkness-has been the subject of controversy for some time. Knapp et al. (1973) measured a gain of about 300 kg per ha during the day, while about the same amount disappeared during the night at the late bud stage, and less than half as much at about early bloom. Starch and sucrose amounted to 70% of the weight changes. Dry matter losses tended to be greater on warm nights, but the relative proportions lost through respiration and transpiration were not determined, Tracer studies by Hodgkinson and Veale (1966) showed that assimilated 14C is rapidly translocated in the form of soluble carbohydrate to the stems and, to a greater extent, to the roots, where it is steadily converted into insoluble forms. In the light, however, the formation of insoluble carbohydrate in stems is greater than in the dark, and there is considerable accumulation of insoluble carbohydrate in the leaves, while transfer to the roots is slowed down. Evidently, assimilate which has recently been formed is not used for respiration during the light (Ludwig and Canvin, 1971). The magnitude of the variations in carbohydrate concentrations creates uncertainty concerning the trends which have been observed in other constituents. Changes in nitrogen fractions, although significant, have been reported to be small in relation to pool sizes, and intermediates do not normally accumulate from rate-limiting steps (Youngberg et al., 1972). No day-to-day weather effects were found.

B. TEMPERATURE

Many reports have shown the importance of air temperature on shoot development. The most consistent effect is that of rate of maturation or time to first flower, ranging from more than 40 days at day temperatures of 20°C or below to 20 days or less at day temperatures of 30°C or above (Jensen et al., 1967;

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Nelson and Smith, 1969; Marten, 1970; Smith, 1970b; Pearson and Hunt, 1972c; Smith and Struckmeyer, 1974). The differences appear to be greater up to the bud stage than between the bud stage and first flower (Greenfield and Smith, 1973), and in general are less at high temperatures. At 35"C, inhibition of floral initiation may cause delay in flowering (Pearson and Hunt, 1972~). Maturation is linked with the rate of node formation, and also possibly with node number, as mentioned in Section III,B. Field and Hunt (1974) reported that the Qlo for node formation was 2.03, with nodes being formed every 3.38 days at 15"/1O"C and every 1.19 days at 30"/25"C. Pearson and Hunt (1972~) observed a similar trend, with a slightly slower rate of 35"/30", attributed to possible water stress. They also found that the rate was faster for regrowth of seedlings than for the primary growth. The effect is paralleled by the more rapid rate of leaf expansion and maturation as the temperature is increased (Wolf and Blaser, 1971b). Dry matter yields at first flower are higher as a rule at lower temperatures because of the extended growing period (Nelson and Smith, 1969; Marten, 1970; Smith, 1970b; Lee and Smith, 1972b; Smith and Struckmeyer, 1974). Growth rates appear to be highest when daylight temperatures are in the region of 20"-25"C (Smith, 1970b; Ueno and Smith, 1970a; Guy ef al., 1971; Bula, 1972). The preceding experiments were all carried out under controlled environment conditions. In the field, Evenson and Rumbaugh (1972) reported that when wheat straw mulch was applied, soil temperatures were lowered by as much as 9"C, plant maturity was delayed, and yields increased by more than lo%, under conditions where it was considered that reradiation and soil moisture differences were of no importance. Plant height, and therefore internode length, do not appear to be greatly affected by temperature (Cowett and Sprague, 1962; Nelson and Smith, 1969), although there are indications that plants in cool regimes tend to be slightly taller than those in warm to hot conditions (Smith, 1970b; Ueno and Smith, 1970a; Vough and Marten, 1971). Although stem and leaf weight and leaf area are all increased at low temperatures, stem growth is proportionately greater than at higher temperatures. Nelson and Smith (1969) found that the total yield at 18"/1O"C was three times that at 32"/24"C, while the leaf weight was only twice as much. Marten (1970) reported that the 1eaf:stem ratio at first bloom was lower at 16"/10"C than at 27"/2 1°C. Stem diameter is greater under cooler conditions (Vough and Marten, 1971), and the weight of stem per unit length of internode declines at an increasing rate as the temperature rises (Field and Hunt, 1974). Leaf area at full expansion is greatly influenced by temperature, reaching a maximum in the vicinity of 20". It decreases gradually as the temperature is lowered (Wolf and Blaser, 1971b; Sato, 1974), and more rapidly as the tempera-

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ture is raised (Nelson and Smith, 1969; Wolf and Blaser, 1971b; Bula, 1972; Pearson and Hunt, 1972c; Sato, 1974). The rate of increase in leaf area is equivalent to the area at full expansion divided by the rate of leaf appearance, and there is some evidence to suggest that this is highest at intermediate temperatures (Nelson and Smith, 1969; Wolf and Blaser, 1971b; Sato, 1974). SLW was reported by Smith and Struckmeyer (1974) to be almost twice as great at first flower under a 21"/12"C regime as compared with 32"/24"C, and this was associated with starch concentrations of 40% and 6%, respectively. Microscopic examination showed that under cooler conditions, chloroplasts accumulated so much starch that cell lumina were not discernible. Leaflets had highly thickened sclerenchyma and phloem cell walls, and were 30% thicker than leaflets at the high temperature regime, with more compact palisade and spongy parenchyma cells. When expressed on a starch-free basis, SLW showed much less variation with treatment. Sato (1974) also found that SLW, leaf thickness, and mesophyll thickness were all greater at 15°/100C than at higher temperatures, while palisade cell diameter decreased steadily with temperature. Stomata1 and epidermal cell densities were lowest and intercellular volume greatest at 20'11 5°C. In contrast, Bula (1972) observed a tendency in three different varieties for SLW to increase from 25" to 35°C. Leaflets grown at 20" and 25°C had larger cells, particularly in the xylem and bundle sheath parenchyma, and more intercellular spaces. There were no obvious trends in leaf thickness. Pearson and Hunt (1972d) found that SLW was lower at 20°/15"C than at 30"/25"C, while Field and Hunt (1974) reported that the decline in SLW was rapid between 15"/lO"C and 20"/15"C, but became more gradual as the temperature increased, with little difference between 25"/20"C and 30"/25"C. These results indicate that starch accumulation is mainly responsible for changes in SLW below about 20°C but that at higher temperatures different rates of cell division and development modify the leaf anatomy, altering cell size, the proportion of void space, and also possibly the dry matter content. It also appears from measurement of carbohydrate levels that leaflets are just as able to adapt themselves to changes in temperature (Greenfield and Smith, 1973) as they are to changes in light intensity. The temperature coefficient for photosynthesis is close to unity over the normal range (Thomas and Hill, 1949; Stanhill, 1962; Pearson and Hunt, 1972a), with a rapid decline below 5°C and above 30°C (Murata et al., 1965). However, when Pearson and Hunt (1972b) raised the temperature in steps over the day from 10" to 40"C, they found that net carbon intake decreased from 20 to about 5 mg per dm2 per hour, suggesting that treatment interactions of some kind may have been involved. Pearson and Hunt ( 1 9 7 2 ~ )found that the root:shoot ratio of seedlings increased more rapidly with time as the temperature was raised, but that the

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asymptotic value finally reached was lower at the time of 50% flowering. The nature of the curves indicated a possible reason for inconsistencies among other results obtained at different stages of growth. However, Smith (1970b) reported that, as temperature increased, plants harvested at first flower had lower total yields but higher root:shoot ratios. It is usually assumed that in a controlled environment chamber, soil temperature follows air temperature, with a lag period depending on the size of the container. However, such factors as radiation level, air movement, and soil moisture content can produce large temperature gradients in the region of greatest root density close to the surface. Using controlled soil temperatures, with air temperatures fluctuating between 15°C and 32"C, Heinrichs and Nielsen (1966) found for a wide range of cultivars that herbage growth was higher at a root zone temperature of 27°C than at lower temperatures, whereas root growth was greater at 12°C than at higher temperatures, and much greater than at 5°C. Despite considerable temperature X variety interactions, the fmdings are consistent with other indications that while fairly low temperatures are suitable for root growth because of reduced respiration requirements, top growth i s restricted as a result of inadequate supplies of nutrients and possibly of water. It seems that root temperature has no appreciable effect on shoot maturation (Nielsen et al., 1960; Heinrichs and Nielsen, 1966; Jensen et al., 1967). Dermine et al. (1967) found that whereas a change from 15.6'/4.4"C to 26.7"/15.6"C resulted in an immediate shoot growth response, the reverse change took 2-3 weeks for growth to slow down, with no comparable reduction in root growth. This could be interpreted as the result of reduced mineral uptake at the lower temperature regime, with a depletion of the reverse already present. The effect of variation between day and night temperatures has received little attention. Steinke (1968) found no significant differences in yield between plants grown at 18"/1OoC and 18"/4"C. Robison and Massengale (1969) concluded that high night temperatures might have been partly responsible for a decline in vegetative growth, carbohydrate reserves, and plant vigor, but other environmental differences between plants grown in the greenhouse and in the field may well have had greater effects. Smith and Struckmeyer (1974) found that plants grown at 3Oo/3O0C not only had yields similar to those at 32"/24"C but were higher in leaf carbohydrate content. Pearson and Hunt (1972a) inferred from their results that translocation from shoots to roots during darkness may have been less at higher temperatures, but more direct evidence would be valuable, Dark respiration increases almost linearly with temperature, corresponding roughly to a Q l o of 2.0 over the lower part of the range (Thomas and Hill, 1949; Murata et al., 1965; Pearson and Hunt, 1972b). There is evidence of possible acclimation (West and Prine, 1960; Pearson and Hunt, 1972b), al-

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though its significance is not clear. Comparing successive harvests in the field over an entire season, Delaney et al. (1974) found an inverse relationship between temperature and leaf respiration, which they suggested might be due to lack of substrate during the summer months. However, the results may be associated with the rate of growth and degree of maturation of the leaves at the time of harvesting. Although respiration is often stilI thought of as wasteful dissipation of assimilate, it is in fact concerned with two vital processes: the repair and maintenance of existing tissues and the provision of energy for synthesis and growth. The deleterious effects of exposure to high atmospheric temperature have been demonstrated by Pulgar and Laude (1974). Plants subjected to treatments of 52°C for 2.5 hours or 46°C for 6.5 hours showed reductions in shoot number and height within 7 days. The effect persisted during the next regrowth period, and shoot lengths were 20% lower than in the controls even at 70 days after treatment. Although roots were not visibly damaged, root dry weights were slightly reduced.

C . WATER

I . Root Growth Plant roots will not grow for any appreciable distance into dry soil, and the extent to which they develop in moist soils depends on the supply of assimilate. Janson (1975b) showed that, in a climate subject to drought, herbage yield, root weight, and root depth during the establishment year were linearly related to the amount of irrigation water, irrespective of time, frequency, or rate of application. The results indicate first that in the coarse free-draining shingle used in this trial, root growth is stimulated by moist, not dry, conditions, and second that the plant is able to take full advantage of water supplies as they become available. With established plants grown in a fine sandy loam of bulk density 1.61, Bennett and Doss (1960) obtained no consistent relation between root weight and soil moisture, but found that effective rooting depth was greater at low soil moisture levels. It is evident that no simple generalization is possible, and that such factors as soil penetrability and water-holding capacity must be taken into consideration.

2. Water Uptake In considering water extraction by the plant, it is useful to start from the premise that uptake from the soil is directly proportional to the root density and to the difference in water potential between root xylem and soil in any given

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region (Bahrani and Taylor, 1961;Kohl and Kolar, 1976). In addition, soil water conductivity may be expected to become limiting at low water potentials. There remains considerable doubt concerning the measured values of plant water potential, and it has been recently reported that levels below -20 bar may commonly occur in alfalfa (Cary and Wright, 1971; Kohl and Kolar, 1976). In fine textured soils, the great majority of the roots are in the top 6-12 inches, in the best position to encounter incoming water from rainfall and irrigation. It is therefore usually observed that soil water in this region is removed much more rapidly than at greater depths (Bennett and DOSS,1963; Lucey and Tesar, 1955). Alfalfa is able to deplete the surface layers just as rapidly as other species, but because of its deeper rooting system it extracts water from lower levels during dry periods (Chamblee, 1958b; Van Riper, 1964). In sandy soils, water is likely to be more evenly extracted at all depths. Under moderate to high saline conditions, alfalfa removes water at depth at tensions well below -15 bar (Brun and Worcester, 1975). At low temperatures, water uptake may become limiting. Ehrler (1 963) found that, compared with rates above 20"C, water absorption was reduced by 20% at 10" and by 70% at 5°C. There were no interactions between contrasting varieties and temperatures. 3. Plant Growth and Water Use

If water uptake were the only consideration, plant growth would be greatest when the soil was at field capacity (100% water availability), but would show little reduction until the soil water potential reached a value of about -2 bar (Kemper and Amemiya, 1957). Since this figure may correspond to less than 25% water availability in sand and more than 75% in clay, it is hardly surprising that trials in which stands have been irrigated at various levels of soil water availability have yielded such different results (Bourget and Carson, 1962; Hobbs et al., 1963; Bezeau and Sonmor, 1964; Peyremorte et al., 1971). The additional complication of excess water supply will be discussed later. Alfalfa has acquired the reputation of being an extravagant consumer of water, despite considerable evidence that this is an unbalanced verdict. Trials in a number of regions have shown that daily consumptive use is similar to that of other crops in which full ground cover is established (Fredricksen, 1938; Halkias et al., 1955; Krogman and Lutwick, 1961; Peck et aL, 1958; Bennett and Doss, 1963; Szeicz et aL, 1969). Various workers have concluded that annual water use depends not so much on species as on length of growing season, proportion of ground cover, rooting depth, and crop yield (Chamblee, 1958b;Krogman and Lutwick, 1961; Sonmor, 1963; Tadmor et al., 1966). During the dry summer months, water consumption by actively growing alfalfa may be similar to that of dormant or semidormant species (Snaydon, 1972~).

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

20 1

During the spring, water demands are comparatively low, and it is usually only during periods when potential evaporation rates are high and growth rates low that water losses become excessive. Yields often decline with successive harvests during the season to a much greater extent than evapotranspiration, with water requirement (weight of water used per weight of dry matter produced) being almost doubled (Cohen and Strickling, 1968; Vorhees and Holt, 1969; Daigger et al., 1970). Hanson (1967) reported that both yields and water-use efficiencies were higher on frequently irrigated plots, and that consumptive use was greatest when irrigation was delayed until essentially all moisture had been depleted to a depth of 6-12 inches. Gifford and Jensen (1967) found that water-use efficiency declined at lower soil water availability, but Lucey and Tesar (1965) reported that it was independent of irrigation regime. High evaporative demand alone does not seem to be responsible for wastage, since Tadmor et al. (1966) showed that under arid conditions, water requirement was 820 in one year and 740 in the next, compared with the mean of 859 established by Schantz and Piemeisel (1927). The general impression is therefore that alfalfa uses water wastefully because it continues to function during periods when water stress restricts its growth and when other plants remain dormant, neither growing nor using water. This places the crop at a further disadvantage. In summer, the deep green mass of an alfalfa field often presents the appearance of an oasis among the surrounding areas, and for this reason becomes subject to the “oasis effect.” Advective winds brought across dry, bare ground supply latent heat flux energy to the crop, increasing evapotranspiration rates by up to 40% (Blad and Rosenberg, 1974), or even more (Hudson, 1965; de La Sayette, 1967). Where moisture supply is adequate and advection is absent, evapotranspiration from a crop which has established full ground cover is close to potential evaporation as governed by meterological factors, particularly incoming radiation (Jackson, 1960; Nicholaichuk, 1964; Krogman and Hobbs, 1965; Hobbs and Krogman, 1966). As the soil dries out, evaporation rates decline according to the gap between the transpirational demand arid the water resources available to the root system. Van Bavel (1967) found that stomata1 conductance started to decrease when the soil water potential reached -4 bar. At -11 bar, evapotranspiration was less than 0.2 of potential evaporation and was controlled by the plant such that it did not exceed 20 mm per hour, regardless of demand; at this stage, the crop was emitting heat to the atmosphere. Numerous trials have shown that evapotranspiration is least in the week following defoliation, despite the higher daytime temperatures above bare ground, and is usually one-quarter to one-half of the rate at full bloom. Where plants remain dormant following harvest during summer drought, water use may be as low as 0.1-0.3 mm per day (Tadmor e l al., 1966). Transpiration increases not only as full ground cover is attained (Krogman and Hobbs, 1965) but also as

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crop height increases, exposing greater leaf surface area and creating greater air turbulence by increasing the aerodynamic roughness of the canopy. The evapotranspiration rate from a tall crop can be twice that from a short one (Hafeez and Hudson, 1965; Grassi and Chambouleyron, 1965). A stand cut weekly is likely to produce somewhat less dry matter than one allowed to reach maturity, but may use only half as much water (Sprague and Graber, 1938). Water use becomes greatest at full flower and during seed formation and ripening (Ktidrev et al., 1970), and it is during this time that differences between cultivars in water requirement become most apparent (Cole e t a l , 1970). Genotypes within cultivars show considerable differences in water-use efficiency (Cole and Dobrenz, 1970), which is associated with htgh forage production, particularly of stems, but is not related to palisade cell density or leaf thickness (Dobrenz et al., 1971). 4. Water Deficit

Bauman (1957) distinguished five phases in moisture stress effects on growth, related to the osmotic potential of the plant during a drying cycle: above -10 bar, optimal growth; -10 to -12 bar, little effect on growth; -12 to -17 bar, growth very slow; -17 to -32 bar, no growth; and below -32 bar, dry weight loss. Stem growth is most affected by water deficit; stems per plant, internodes per stem, internode length, and branching are all reduced (Perry and Larson, 1974), and eventually elongation ceases (Lucey and Tesar, 1965). Under mild deficits, the lower yields may be of higher quality than in plants with adequate soil moisture (Jensen et a l , 1967), but further desiccation leads to general plant deterioration. Shoot growth appears to be slightly more affected than root growth (Bourget and Carson, 1962; Janson, 1975b), presumably because of the water potential gradient within the plant, and growth appears to be diverted to carbohydrate storage in the root (Willard, 1951 ; Cohen et al., 1972). Striking changes in leaf morphology under arid conditions have been described by Gindel (1968). Mean leaflet size was only 1 cm2 during the dry season, compared with 4 cmz at the end of the wet season. Stomate and epidermal cell densities were somewhat greater in the smaller leaves, but it appeared that cell expansion was reduced more than cell division. Small cells, having proportionately less volume reduction when desiccated, and high negative osmotic values are characteristic of drought-hardy plants (Russell, 1959). Alfalfa has no more ability than many other plants to remove water from soil before permanent wilting occurs (Briggs and Shantz, 1912). Murata et al. (1966) found that respiration was reduced when the soil water content was reduced to 45%, photosynthesis at 35%, and leaf water content at 25%. All three values

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

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were reduced by about 40% at a soil water potential of -4.5 bars. While these soil water availabilities cannot be interpreted in terms of plant water status, they do suggest that vapor loss may continue long after growth has been brought to a halt by insufficient turgor. Loper (1972) reported relative turgidities of less than 50% in wilted alfalfa leaves, and it is difficult to see in what way this could be of benefit to the plant. It does not appear to be characteristic of other drought-resistant species (Sanchez-Diaz and Kramer, 1973). Further studies are required of this most important and inexplicably neglected aspect of alfalfa physiology. Night opening of stomata under arid conditions was observed by Loftfield (1921), but no explanation for its occurrence in alfalfa has been put forward, although it is familiar in plants with crassulacean acid metabolism. It can result in measurable transpiration losses, particularly following hot days, but the effect on the total water economy of the plant is likely to be small (England, 1963; Van Bavel, 1966), except under conditions of severe advection (Abdel-Aziz et al., 1964; Rosenberg, 1969).

5. Excess Water Alfalfa is highly susceptible to waterlogging (Finn e t al., 1961). Differences in tolerance between cultivars have been reported (Rogers, 1974). The initial effects are usually attributed to lack of oxygen in the root zone, resulting in the formation of toxic substances which produce root xylem necrosis and yellowing and wilting of leaves. Symptoms appear sooner and with greater severity as the root zone temperature increases (Erwin et al., 1959; Finn e t al., 1961; Heinrichs, 1972; Cameron, 1973), presumably since higher root respiration rates deplete the oxygen supply more rapidly; air temperature has no effect (Cameron, 1973). Damage is greatest in recently defoliated plants (Erwin et aL, 1959; Rai et aL, 1971; Cameron, 1973), which is probably due to the small amount of leaf area available for gas exchange and to a lack of root activity. The more transpiration is restricted, the longer the plant takes to obtain relief from the conditions causing the problem. In seedlings, excess soil water can also lead to subsequent impairment in mineral absorption (Andrew, 1966). Waterlogging may induce manganese toxicity in certain soil types, with possible lasting effects (Graven et al., 1965). Primary symptoms are apparently not associated with pathogens (Cameron, 1973), but wet soils enable Phytophthora oospores to germinate and release zoospores that infect the alfalfa root (Lueschen et al., 1976). Frosheiser and Barnes (1973) found that Medicago sativa, M. falcata, and M. lupulina were the only forage legumes susceptible to Phytophthora megasperma in the field, and they concluded that alfalfa can tolerate wet soils in the absence of plant pathogens. But although infection ceases when the soil dries out, tap roots may

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be partly or completely rotted off, and the shallow adventitious lateral shoots which subsequently develop are usually inadequate to restore full plant vigor (Lueschen et al., 1976). In the seeding year, Phytophthora may increase in incidence and severity because of stress factors such as high soil moisture, frequent cutting, and high seeding rates (Pulli and Tesar, 1975). Wahab and Chamblee (1972) found that irrigation at 50% water availability initially increased yields, but eventually reduced them, with loss of the stand. The time taken for damage due to various organisms to become evident vaned from the first harvest to 2 years, depending on variety. Hobbs et al. (1963) reported that irrigation at 75% water availability gave higher yields than at lower levels only for first-year stands. Lehman et aZ. (1968) carried out experiments on two different soil types which indicated that the effect of irrigation on yield depended greatly on soil water permeability and subsurface drainage. The results suggest that rate of irrigation may have even more important consequences than timing, and since the rooting depth will often closely correspond with depth of infiltration, the range of optimal water supply may lie within fairly narrow limits. I). MINERALS

1. Root Growth Root penetration depends on the nutrient supply at each level. Roots grow vigorously in fine sand or silt where phosphorus is available but do not move into regions of moist gravel or coarse sand, even when phosphorus fertilizer is applied (Fox and Lipps, 1955a). There is a lack of nodulation and root branching in phosphorus-deficient soils (Fox and Lipps, 1955b). In strongly acid soils, the taproot grows down to the limed-unlimed interface and stops abruptly, sending out a number of lateral roots (Buss et al., 1975b). At slightly higher pH levels, taproots do not develop normally, and there is less proliferation of fibrous roots (Pohlman, 1946; Schmehl et al., 1952; Moschler et al., 1960). Liming at depth can stimulate root growth even when the surface layers have been limed (Pohlman, 1946). On the other hand, when calcium is obtained from lower depths, nodules may develop on roots in the acid topsoil (Fox and Lipps, 1955b). In addition to phosphorus and calcium, boron is also needed for the growth of fine roots (Simpson and Lipsett, 1973).

2. Uptake by Roots At low temperatures, mineralization and rates of equilibration of sulfur and phosphorus in the soil are greatly reduced, and responses to fertilizer application

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205

are more likely to be obtained (Jones, 1970; Sutton, 1969). Restricted water absorption by roots below 10°C may also limit mineral intake. Phosphorus content of alfalfa herbage has been shown to increase with temperature, with accompanying effects on yield (Nielsen et al., 1960; Parsons and Davis, 1960; Levesque and Ketcheson, 1963; Heinrichs and Nielsen, 1966). With potassium, the evidence is less clear. Nielsen et al. (1960) found that uptake was proportional to growth and that herbage concentration at final harvest showed no definite trends with root temperature. D. Smith (1971) reported low herbage concentrations and deficiency symptoms at cooler temperatures, but herbage dry weight was correspondingly higher. Plant calcium and magnesium levels show a complementary decline as temperature increases (Nielsen et al., 1960; D. Smith, 1970a, 1971). Soil moisture stress may limit the uptake of phosphorus, Several workers (Kilmer et al., 1960; Younis et al., 1963; Snaydon, 1972b) have reported that the phosphorus content of herbage increased linearly with soil water availability, although Bourget and Carson (1962) found no effect of water regime. Although phosphorus absorption may be influenced by soil moisture, it need not parallel water absorption. Lipps et al. (1957) reported that even where a water table was present, phosphorus was not removed from the subsoil when surface soil moisture was high. Little uptake may occur from the drier region between the surface and the region of capillary rise (Lipps and Fox, 1956; Campbell et al., 1960; Simpson and Lipsett, 1973). In many circumstances, it may be difficult t o tell whether growth is restricted by phosphorus or water supply. Sorensen et al. (1968) suggested that high levels of sulfur might accumulate in the plant under water restriction. Kilmer et al. (1960) did not find that growth response to additional water supply was accompanied by increases in the herbage content of minerals other than phosphorus. However, boron deficiency can be induced in plants of high boron content by a sharp reduction in soil moisture (Buss et al., 1975a), since the roots cannot absorb the element from dry soil, even if moisture is present in a deficient subsoil (Hobbs and Bertramson, 1950). The detrimental effects of low pH, apart from those on nodulation, are attributed to aluminum and manganese toxicity. Alfalfa is highly susceptible to aluminum toxicity (Andrew et al., 1973), which may be related to its high cation exchange capacity (Vose and Randall, 1962). Compared with other temperate legumes, alfalfa is highly inefficient at removing calcium from deficient soils, although appreciable amounts can be taken up when the deficiency is not extreme because of the extensive root system (Andrew and Norris, 1961). The beneficial effects of adding lime on base saturation, soil exchangeable aluminum and manganese levels, and herbage yields are highly correlated (Moschler ei a f , 1960; Munns, 1965; Hutchinson and Hunter, 1970; Helyar and Anderson, 1971; John etal., 1972; Buss et al., 1975b; Janghorbani et al., 1975).

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The range of pH over which amelioration occurs is naturally dependent on soil characteristics, and when both high acidity and low phosphorus availability are encountered, interactions between lime and phosphate applications are to be expected (Munns, 1965; Janghorbani et al., 1975). Excessive liming not only may have little beneficial effect, but also may induce deficiencies of boron (Mannetje, 1967) or manganese (Bear and Wallace, 1950). Visual symptoms of aluminum toxicity in shoots resemble those of phosphorus deficiency, while those in roots show similarities with those of calcium deficiency (Andrew and Vanden Berg, 1973). Toxicity is associated with high concentrations of aluminum or manganese in the tops, rather than in the roots (Ouelette and Dessureaux, 1958; MacLeod and Jackson, 1964; John et al., 1972; Andrew er aZ., 1973), and is evidently overcome by the uptake of calcium in sufficient quantities to reduce the ionic concentration of the toxic element to the point where phosphorus is not precipitated or otherwise immobilized in the roots. Manganese ions can also reach toxic levels in the soil either when extremely hot, dry conditions prevent oxidation to unavailable compounds or when waterlogging causes reductions of higher oxides by anaerobic bacteria (Graven et al., 1965; Simon er aL, 1974). 3. Nitrogen Fixation Nitrogenase activity of nodules on Medicago sativa roots is high between 2" and 38OC, with a maximum at 35°C (Dart and Day, 1971), and Rhizobium melibti is reported to be capable of withstanding high temperatures (Brockwell and Phillips, 1964). However, nitrogen furation and assimilation and total yield may be greatly reduced when soil temperatures of 40°C are frequently exceeded (Rogers, 1969), and Godley (1968) reported that the frequency of functional nodules was impaired above 27°C. At low temperatures, it has been suggested (Leach, 1968b) that winter-active varieties may be capable of faster growth than the symbiotic source can sustain. Water deficits may reduce the frequency and duration of functional nodules (Codley, 1968), and have major effects on nitrogen fixation (Imangaziev and Patakhov, 1968). Lack of aeration leads to abnormal functioning of nodules. BeIow pH 5.5, root hairs are prevented from curling and becoming infected. The initiation of infection is the most calcium-dependent and acid-sensitive stage of nodulation, and once it is completed, a pH of 4.4 does not hamper further development (Munns, 1968). Other effects are likely to be direct consequences of failure in carbohydrate supply by the host plant. Shading depresses nodulation proportionately more than root growth, and nodules which are formed may not be functional (Pritchett and Nelson, 1951; McKee, 1962). Clipping is reported to have little

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effect on nodulation (Godley, 1968), although repeated short cutting may result in weak root development with few and discolored nodules (Langer and Steinke, 1965). The loss of transient roots, whether from drought, flowering, winterkilling or other causes must lead to the decay of the attached nodules, the contents of which are liberated into the soil, where they would be readily available for further cycling (Holford, 1968). The quantities involved may be of little consequence, since fmation products appear to be transferred immediately to the host plants, and the roots and nodules do not act as substantial storage organs (Pate, 1958). Alfalfa leaflets have a high nitrate reductase activity at young vegetative stages, indicating their ability to supplement fixation with soil nitrogen when demands are high (Eskew et al., 1973). However, additional nitrogen supply does not fully compensate for light restriction and tends to inhibit nodule development (Schertz and Miller, 1972), even to the extent of subsequently inducing symptoms of nitrogen deficiency (Kunelius, 1974). In established stands, increases in yield and stem nitrogen content produced by nitrogen fertilizer application may be too small to be worthwhile (Lee and Smith, 1972a). However, where very large amounts of forage are being removed (over 20 tons per ha per year) supplementary nitrogen may substantially increase yields (Vartha, 1972; Hoglund et al., 1974), suggesting that the supply of symbiotic nitrogen is becoming limiting. Yield responses to applied nitrogen at low temperatures have also been reported (Hamilton, 1970). Plant nitrogen content is reported t o increase with temperature (Heinrichs and Nielsen, 1966; Lee and Smith, 1972b), and the rapid fall in nitrogen content in roots during early spring (Bula and Smith, 1954; Bowren et al., 1969) may also be associated with inadequate fixation. 4. Mineral Interrelationships Plant mineral nutrition is dominated in many respects by nitrogen, and deficiencies in other elements often become manifest through the inability to carry out normal protein synthesis. MacLeod and Suzuki (1967) found changes in the amino acid pool that indicated a shift from nitrogen catabolism to anabolism as the K:N ratio of the nutrient solution increased, with maximum yields at a ratio of unity. Both potassium and sulfur deficiencies increased the aspartic acid:glutamic acid ratio, but produced contrasting changes in other amino acids (Adams and Sheard, 1966). The buildup of arginine and asparagine in sulfur and phosphorus deficiencies suggests alternative nitrogen storage when further synthesis is blocked (Mertz et al., 1952; Gleiter and Parker, 1957). From another aspect, the direct effect of potassium upon photosynthetic activity (Cooper et al., 1967) may be sufficient to account for most of the observed increases in leaf area expansion, persistence of lower leaves, stem

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growth (D. Smith, 1971), and root carbohydrate storage (Matches et al., 1963; MacLeod, 1965b; Reid et aZ., 1965). Higher leafstem ratios are to be found in plants whose growth is restricted by a deficiency of potassium (Bear and Wallace, 1950) or of phosphorus (Gleiter and Parker, 1957), and nitrogen values are therefore likely to be higher than in normal plants. The impairment of nitrogen assimilation by mineral deficiencies will have the opposite effect, however, and nitrogen concentrations have been found to be slightly lower in sulfur deficiency (Caldwell et al., 1969). The ionic environment of the plant root can seldom be evaluated satisfactorily by extraction procedures, and changes in plant composition are commonly used to interpret nutrient status. However, supplying a deficient element is likely to increase yield without substantially raising plant concentrations (Larson et al., 1952; Andrew and Robins, 1969a,b), while inconsistent yield increases at high potassium levels suggest that either potassium is needed to maintain parity with high nitrogen levels or it is influencing the uptake of some other element. Mineral uptake is largely governed by the tendency to maintain a stable balance between major inorganic cations and anions and by the ionic balance in the soil. Plant concentrations of calcium and potassium, for example, are more dependent on the Ca:K ratio in the soil than on the absolute values (Wallace, 1952), and an increase in one often results in a complementary reduction in the other. Simple anion substitution is probably responsible for increases in plant sulfur and decreases in phosphorus and boron produced by sulfur application (Caldwell et al., 1969). Saline soils have little effect on herbage cation content, since sodium uptake is always low, and chloride is accumulated at the expense of other anions (Brown, 1958). The addition of ions to the soil involves an equivalent number of ions of opposite charge and equal availability, and the extent to which these are taken up by the plant will depend on the ability of ions already present to compete with them. Gervais et al. (1962) found that when responses to both phosphorus and potassium were obtained, the addition of either to the soil produced a reduction in plant concentration of the other. The results of D. Smith (1971) are of interest, since they show that the application of potassium chloride or sulfate resulted in much lower phosphorus levels, particularly at low temperatures. Further progress in evaluating nutrient requirements might result most rapidly from a realization of the need for more comprehensive plant analysis and systematic examination of the results, viewed in the context of quite basic considerations. Nyatsanga and Pierre (1973) pointed out that although legumes obtain most of their nitrogen from the air, bases are derived from the soil, and they calculated that an annual yield of 10 tons per ha would produce soil acidity equivalent to 600 kg per ha2 of CaC03. In a greenhouse experiment, they

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

209

measured an amount of nitrogen fixed by alfalfa over 167 days sufficient to lower the soil pH from about 5.8 to about 4.9. The result has implications concerning the soil environment and plant growth which require further investigation.

VI.

Phases in Development

A. BUD AND SHOOT INITIATION

In cold-tolerant varieties, dormancy of crown buds is evidently established at the time of physiological changes associated with the hardening process (Grandfield, 1943), and the next season's growth originates from these overwintering buds as well as from buds developing during the early spring (Nelson and Smith, 1968). In winter-active types, buds continue to develop at colder temperatures, although at a reduced rate, and they are vulnerable to frosts and grazing. During the growing season, buds commonly appear at the base of the plant at about the time when the first floral buds become visible (Nelson and Smith, 1968; Janson, 1975a). If the plants are left uncut, elongation starts at about full bloom, with the old stems subsequently lodging and drying. Both the size and the number of basal shoots increase as the plant enters the flowering stage (Cowett and Sprague, 1962; Leach, 1968b; Janson, 1975a). The appearance of new shoots is often regarded as a sign of herbage maturity, even where growth is continuous and flowering sporadic, as in the cool Andean regions (Crowder et aZ., 1960). However, Bartels (1956) in Victoria, Australia, observed a wide distribution of shoot lengths, with little indication of distinct growth cycles; no information was given to indicate what environmental factors may have been involved. New crown growth can also be induced by restraining the older shoots in a horizontal position close to the ground (Tysdal, 1946). The earlier cutting is carried out, the more gradual is the start of bud elongation and the higher the proportion of shoots originating from the crown (Leach, 1968a), and shading plants before defoliation further delays the start of growth (Leach, 1969). Once growth starts, rates of elongation and weight gain are largely independent of cutting treatment (Leach, 1968a). If sufficient stubble leaf remains, photosynthetic activity may be high enough to reduce carbohydrate mobilization from the roots (Hodgkinson, 1973), and crown temperatures may be lowered somewhat (West and Prine, 1960), but little regrowth is likely to originate from stubble nodes more than an inch or so above the crown (Leach, 1970a). Growth is initially more rapid at higher temperatures (Leach, 1971a), but exposures to air temperatures above 45" reduces shoot numbers (Pulgar and Laude, 1974).

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The number of stems that develop after defoliation is correlated with crown width, weight of previous harvest, and competition from neighboring plants (Miller et al., 1969), a finding which is consistent with the conclusion of Cowett and Sprague (1962) that environmental factors affect tillering indirectly through their effects on plant growth and vigor. B. ROOT CARBOHYDRATE STORAGE

Although an association obviously exists between shoot growth initiation and root carbohydrate storage, causal relationships have yet to be unequivocally established. Adequate root carbohydrate levels are not in themselves responsible for shoot initiation, although they evidently have an important bearing on the success of subsequent growth. Chatterton et al. (1974) found that the dry weights and total nonstructural carbohydrate (TNC) concentrations in herbage, crowns, and roots of plants which tillered and flowered early were higher than those in plants which matured later. Cowett and Sprague (1962) observed that even where roots were small as a result of moisture stress, yield and stem number were comparable with those of unstressed plants when grown at adequate levels of soil moisture. In contrast, Ueno and Smith (1970b) found that stem number was directly related to root and crown weight at harvest. They also found that the proportion of TNC lost from the roots was similar for plants of different root size. Quite possibly the respiration requirements of the extensive root system are at least partly responsible for the greater dependence of alfalfa on these reserves than is true of other species. The effects of temperature on rates of depletion and renewal of carbohydrate in the roots reflect those on shoot maturation. The fall in concentration is more rapid at higher temperatures (Singh and Winch, 1974), but the leaf area needed to restore self-sufficiency is also attained earlier (Silva, 1968). The subsequent peak in concentration is reached at about the time of first flower, which is much earlier at high temperatures, but the level is likely to be lower than in cooler conditions (Nelson and Smith, 1969). Moderate shading may make little impression on root carbohydrate concentrations (Matches et al., 1963), but low light levels accentuate the effect of severe and repeated defoliations (Steinke, 1968). Very little root growth occurs in plants grown in the dark (Smith and Silva, 1969). Prolonged dry periods lead to virtual dormancy of shoots and crown buds, and grazing at such times is unlikely to seriously harm subsequent production (Wilman, 1965; McAuliffe, 1967). However, Snaydon (1972a) reported that irrigation water applied at the rate of 5 mm every 8 days gave higher yields during a dry summer period, but lower yields in the following autumn, than the same total amount applied in larger aliquots less frequently. Since such small

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

21 1

additions could hardly have made any difference in the soil moisture content, he suggested that each watering might have stimulated translocation from the taproot. Cohen et al. (1 972) found that when plants were irrigated at the time of cutting, the rate of regrowth was twice as great as when irrigation was withheld for 10 days, but the fall in TNC was 8 times as great. Other evidence of a more general nature suggests that alfalfa may not be well suited to moist environments. Willard (1951) stated that root weights can be twice as great at the end of a dry year as following a wet season, and that in dry regions alfalfa could be cut at much earlier stages than in humid regions. Unfortunately, the distinction between humid and high-rainfall climates is not always made, and no experiments to determine the effects of atmospheric moisture on alfalfa growth have been described. Reports have indicated the difficulty in maintaining stands in such diverse high-rainfall areas as Florida (Prine, 1966), Hawaii (Wilsie and Takahashi, 1937), and Ireland (Farragher, 1969). Such factors as waterlogging and disease may of course contribute to lack of persistence; but the results of Weir et al. (1960) in California, for example, indicate that frequent cuttings with the aid of irrigation in a semiarid climate cause no lasting damage to plant stands. Willard (1951) also noted that, in humid regions, shoots very seldom appear uniformly, and may be present before full bloom. At the low radiation levels experienced beneath a stand of high leaf area index, it would appear that vigorously growing new shoots must be parasitic and in competition with the roots for assimilate. Whether these shoots eventually died or were mowed before they developed fully, they would contribute little to total yields, but might well constitute a continuing drain on plant reserves.

C. REGROWTH CHARACTERISTICS

As might be expected from general seasonal trends, regrowths display a number of features observed in controlled eiivironment studies at elevated temperatures. Regrowths are in general shorter and take less time to reach maturity than the first growth (Dent, 1959). They usually have a higher leafstem ratio, and the stem diameter is reported to decline with each cut (Mowat et al., 1967). However, reports vary as to whether stems become more lignified or less digestible (Lagowski et al., 1958; Jensen el al., 1967; Mowat et al., 1967). Rates of accumulation of dry matter and leaf area index may be either slower than for the first growth (Hunt et al., 1970), or comparable, but with a lower maximum (Greub and Wedin, 1971). The decline and recovery in acid-hydrolyzable carbohydrate concentrations occur more rapidly with each successive cutting, corresponding with rates of floral initiation (Dobrenz and Massengale, 1966).

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D. FLOWER AND SEED FORMATION

Insufficient information is available to show whether any clear relationship exists between floral and vegetative development. Maturation and flowering take place more rapidly as the temperature is increased (Nittler and Kenny, 1964; Sato, 1971; Section V,B), and plants may even fail to flower at temperatures around 20°C (Roberts and Struckmeyer, 1939; Sato, 1971). Flowering is promoted at greater day lengths, and is earliest and most prolific under continuous light (Nittler and Kenny, 1964; Murray, 1967; Guy et al., 1971); although the effect is primarily photoperiodic, higher plant growth rates may also contribute. The detrimental effect of low light intensities (Nittler and Kenny, 1964) is probably a direct consequence of reduced plant vigor. However, the mechanism of induction at low temperatures (Roberts and Struckmeyer, 1939) remains obscure. Interactions between pollinating insect, plant, and environment are of great importance, but not well understood (Heinrichs, 1965; Kauffeld et al., 1969). High temperatures may increase the number of inflorescences pler plant but reduce the number of flowers on each inflorescence (Guy et al., 1971), while high night temperatures are reported to cause flowers to abscise (Roberts and Struckmeyer, 1939). In comparisons of soil moisture and spacing treatments, Tysdal (1946) showed that a strong inverse relationship existed between forage and seed production. Soil moisture stress is likely to reduce the number of flowers per plant (Grandfield, 1945; Tysdal, 1946). However, seed production is reported to be maximized by maintaining a soil moisture potential between -2 and -8 bar after the start of blossoming (Taylor et al., 1959), while high humidities can seriously reduce the percentage of flowers setting pods, particularly at temperatures around 27°C (Grandfield, 1945). Lodging also affects the proportion of seeds set, rather than the number of flowers (Tysdal, 1946). It would seem that conditions which favor the elongation of new shoots can reduce the supply of assimilates available for seed formation, and high carbohydrate reserves in the roots may be of value for this reason (Grandfield, 1945). Undei severe stress conditions, such as dry soils or temperatures above 30°C,a high proportion of seeds either abort or do not develop normally (Taylor et al., 1959; Dane and Melton, 1973). The effect of increased spacing depends largely on whether the plants are able to take full advantage of the soil volume and the irradiated area, and whether the additional racemes per stem more than compensate for the smaller number of stems per unit area (Nittler and Kenny, 1964; Dovrat et aL, 1969). If full ground cover is not attained, radiation levels and temperatures are likely to be higher within the canopy, and humidities lower.

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

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E. SEEDING AND THE SEED

Self-seeding rarely occurs to any great extent in alfalfa, and stands usually become thinner and less productive with age. It is therefore of interest that Medicago falcata is reported to increase and become a dominant component of swards in which it is oversown (Rabotnov, 1969). A number of reports have shown that alfalfa is able to regenerate if the stand is rested during mild weather following good rainfalls (Young et al., 1958; Clinton, 1968; Cameron and Mullaly, 1970; Campbell, 1974; Douglas, 1974). Seed produced under cool (6"-21"C) temperatures is reported to be heavier than under hot (16"-32°C) conditions, giving rise to more vigorous seedlings in the early stages of growth (Walter and Jensen, 1970). The microenvironment of the seed is critical for successful germination. Whereas grass radicles tend to enter the soil at angles between 45" and 90", the legume radicle generally grows along the soil surface and enters at obtuse angles, with consequent exposure to desiccation (Campbell and Swain, 1973). The diameter of the root apex is greater than for grasses, and the legume seedling lacks coleorhizal hairs which provide root anchorage to the soil surface. In contrast to grass seeds, entry by secondary roots after the death of the radicle is rarely successful. Consequently, the time for radicle entry is often delayed, and mortality may be high, particularly on smooth or fine structured soils of high bulk density. Establishment is encouraged if sufficient dead vegetation or surface roughness is present to restrain seed movement during penetration (Dowling et al., 1971). Exposure to direct sunlight is clearly to be avoided. Miles (1969) observed that seeds that fell into frost cracks and were covered with soil by later rains germinated well. Since the greatest emergence force is developed within 1-2 days, crusting of the soil surface may prevent penetration (Jensen et al., 1972). Damage to the unprotected emerging cotyledons may cause prolonged reduction in vigor and growth rate (Bignoli, 1950). The temperature requirement for initial development appears to be higher than that for older plants. Pearson and Hunt ( 1 9 7 2 ~ )concluded from their results that optimal growth rates during establishment were highest at 20"/1 5"C, but later shifted to lS"/lO"C. Smoliak et al. (1972) found that the weight and lengths of both tops and roots at 4 weeks from germination increased considerably with each temperature increment from 7" to 27°C. The data of Heinrichs and Nielsen (1966) indicate that the time from seeding to first flower was much greater at a root temperature of 5°C than at higher temperatures, whereas in subsequent cuts, the time to reach maturity was unaffected by root temperature. McElgunn (1973) showed that cold temperatures reduced the rate of germination, while alternating temperatures of 13"/2"C also reduced total germination.

214

K.R. CHRISTIAN VII. Plant Associations

A. 1NTRASPECIFIC:PLANTDENSITY The individual plant perceives its neighbors only as environmental boundaries, regions into which expansion is restrained or curtailed by the lack of light, water, or nutrients. Survival is contingent upon meeting the minimum requirements for viability within these confines, including the readiness to occupy less favorable areas, At the interfaces, its success in direct competition depends on the extent to which it can deplete one or another of the resources to a level below that which can be endured by neighboring specimens. Even in a monospecific sward, wide ranges in plant vigor and physiological tolerance are superimposed on random variations in spacing and soil conditions. The main effect of increased density is a general reduction in size and dry weight of individual plants (Miller et al., 1969; Takasaki et al., 1970; Roufail, 1975). Roots tend to be lighter, of smaller diameter, and with less branching (Hansen and Krueger, 1973; Radei, 1974; Roufail, 1975), while nodulation may be depressed (McKee, 1962). Stems are usually thinner (Mowat et al., 1967) and somewhat shorter (Chisci, 1966), and therefore of lower weight (Takasaki, 1972), with less branching and lower dry matter content (Dovrat et al., 1969). There are fewer stems per plant (Marten et al., 1963; Chisci, 1966; Miller et al., 1969; Roufail, 1975), and presumably less leaves, although the effect of plant density on leaf area has apparently not been studied. In addition, relationships between yield, crown width, and stem length and number alter with plant spacing (Rumbaugh, 1963). Although the seasonal rhythms of production in different varieties are similar for spaced plants and broadcast swards, the relative yields are not comparable (Chisci, 1966). Because of the inverse relationship between plant size and density, correlations between yield and stand density are likely to be unreliable (Ronningen and Hess, 1955; Gross et al., 1958). High seeding rates may increase production in the establishment year by providing more complete ground cover (Moline and Robison, 1971; Takasaki, 1972), but the gaps tend to close up as time goes on (Kramer and Davis, 1949). A number of workers have observed that very different rates of seeding result in similar yields after the establishment year, while plant densities move toward a common lower limit (Marten et al., 1963; Jacquard et aZ., 1967; Takasaki et al., 1970; Scateni, 1972; Takasaki, 1972; Palmer and Wynn-Williams, 1976). The proportion of seeds producing plants declines with seeding rates and, after establishment, deaths are density-dependent; higher seeding rates apparently do not improve persistence (Palmer and Wynn-Williams, 1976). Plants which die are mainly below the mean in height, stem number, and dry weight at previous harvest (Takas&, 1971). It has been suggested (Palmer and Wynn-Williams,

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

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1976) that satisfactory yields could be maintained at 15 plants per m2. According to Chamblee (1958a), most of the competition of alfalfa with itself is above ground, but this might not be the case if water or nutrients become limiting, and on shallow soils, at least, higher densities would probably be needed. B. INTERSPECIFIC COMPETITION

The value of mixed swards lies in complementary growth, but competition is always present. Roberts and Olson (1942) found no cases when both legume and grass benefited or were injured by association, and the root weights of both are likely to be reduced (Aberg et al., 1943; Langille et al., 1965). In a critical review of the literature, O’Connor (1967) concluded that alfalfa-grass mixtures do not provide large margins in yield over the pure legume, except where the grass fills seasonal gaps, although considerations other than those of dry matter production may often be important. Nevertheless, successful maintenance of mixed swards requires a much keener appreciation of the growth patterns and responses of the component species than is needed for a pure stand. Conditions during establishment under a cover crop are often crucial. If soil moisture is inadequate, the cover crop will use it all, and the alfalfa will die from desiccation; Peters (1961) found that oats had a severe effect on establishment during a dry summer. On the other hand, prolonged rain or irrigation may shade the young alfalfa plants for an excessive length of time (Janson and Knight, 1973; Vartha and Allison, 1973). Buxton and Wedin (1970) considered that a suitable cover crop was one such as oats, which was aggressive enough to smother weeds initially, but which had a more open canopy than that of natural weeds. They found that extreme shading, with less than 2% incident radiation at ground level for up to 1 month, did not impair alfalfa survival. Under such conditions, however, the growth of shade-tolerant weeds is encouraged. Klebesadel and Smith (1960) reported that cutting the oats at maturity had little more effect on subsequent alfalfa production than early cutting. Frequent clipping to reduce weed competition is detrimental to the young alfalfa stand, while no clipping at all is even worse (Sprague er al., 1963; Janson, 1971). Provided survival is adequate, the method of establishment has apparently little carry-over effect on subsequent performance (Barker et aL, 1957; Sprague et al., 1963; Hansen and Krueger, 1973; Nordquist and Wicks, 1974). If severe winter conditions prevent growth of the grass species, the grass is more likely to replace lucerne production during the remainder of the year than to augment it (Douglas and Kinder, 1973). Because alfalfa displays little activity at low temperatures it is apt to become smothered by grasses before it starts spring growth. In semi-arid conditions, species which start growth early in the season also tend to become dominant by using all the available soil moisture (Kilcher and Heinrichs, 1966).

216

K. R.CHRISTIAN

Alfalfa competes with tropical legumes for soil moisture (Yates et at., 1971), and during long dry summer periods it may eliminate grasses from the sward by both competing for moisture near the surface and obtaining further supplies at depth (Dann, 1962; Vartha, 1972). But heavy clay soils with waterlogging tendencies encourage the takeover by summer-active grasses (Edye and Haydock, 1967). Spatial variations in mixed-sward microclimate were demonstrated by Larson and Willes (1957) in sowings between 80-inch corn rows running east-west. The soil temperature of the band on the sunlit side of the rows was 4"-7"C higher than on the shady side, and soil moisture was significantly lower. Stand counts of alfalfa increased dramatically from the sunlit side to within 12 inches of the shaded side of the next corn row. At low soil pH, clovers tend to become the dominant legumes, while the addition of lime shifts the balance in favor of alfalfa (Griffith et al., 1966; Helyar and Anderson, 1971). Because the cation exchange capacity of alfalfa is roughly twice that of the highest values among the cultivated monocots, alfalfa is unable to compete strongly for soil potassium (Drake et al., 1952). Alfalfa herbage contains much more calcium and less potassium than associated grass or weed species (Comstock and Law, 1948; Bear and Wallace, 1950; Lawton and Tesar, 1958). The uptake of potassium is lower when competing grasses are present (Coffmdaffer and Burger, 1958; Langdle et al., 1965), and is still further reduced by nitrogen application (MacLeod, 1965a). Accordingly, the addition of potassium is usually essential to maintain alfalfa in the stand, while the necessity to add calcium to reduce soil acidity means that alfalfa is unable to exert any corresponding nutrient restriction on competing plants. Phosphorus is taken up competitively from the topsoil by alfalfa (Massey and Sheard, 1970), although there is some suggestion that phosphorus application favors the growth of other species (Markus and Battle, 1965; Harris et al., 1966). Again, because of its strong root system, alfalfa may gain advantage over its rivals by obtaining access to minerals in short supply near the surface (Blakemore et d.,1969; Jones, 1970). Nitrogen fertilizer invariably leads to a higher proportion of grasses and weeds at the expense of alfalfa (Bear and Wallace, 1950; Gewig and Ahlgren, 1958; Carter and Scholl, 1962; MacLeod, 1965a; Cooke et al., 1968; Peters and Stritzke, 1970; Chan and MacKenzie, 1971; Kust, 1971). Where the legume becomes suppressed, total yields may decline still further because the dominant grass species experiences nitrogen deficiency (Hamilton et a!., 1969). The beneficial influence of alfalfa upon the growth of adjacent plants is evidently dependent on environmental conditions as well as on management practices, but no consistent pattern has emerged (Roberts, 1946; Tewari and Schmid, 1960; Simpson, 1965; Cameron and Mullaly, 1969). Despite the large amounts of

ENVIRONMENTAL EFFECTS ON ALFALFA GROWTH

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nutrients in alfalfa roots, Whitehead (1970) considered that root decomposition would release little nitrogen into the soil because of the hgh C:N ratio, and that little phosphorus or sulfur would be either mineralized or immobilized. Regulating the time intervals between grazings or cuttings affords perhaps the most effective means of controlling pasture composition. Frequent defoliation almost always reduces the proportion of alfalfa in the sward, while the longer it can maintain a closed canopy and dry out the surface soil layers, the better its chances of eliminating competition. Maintaining the vigor of the grass species may depend on its morphological stage of growth at cutting, particularly on whether or not it has reached the heading stage and whether new basal or axillary tillers are available for immediate growth (Parsons, 1958; Smith et al., 1973). Frequent cutting of cocksfoot (Dactylis glornerutu), for example, stimulates tillering and allows rapid recovery and leaf production from the basal storage organs (Williams, 1950; Barker et al., 1957). Yields of grasses relative to that of alfalfa are likely t o be increased by greater stubble height (Wolf et al., 1962). Selective grazing may destroy emerging alfalfa shoots and buds and encourage weed growth, while the effect of urine on pasture composition may depend on the relative amounts of nitrogen and potassium (Whitear et al., 1962; Cuykendall and Marten, 1968).

V I I I.

Genetic Adaotation to Environment

The richness and diversity of the alfalfa gene pool is such that its extension to new areas would seem to depend only on the effort devoted to appropriate breeding programs. Problems involving pests and diseases have been overcome in regions where the value of the crop has already been firmly established and recognized; there is less demand for its adaptation to places where it is now considered either unsuitable or only marginally useful, but the potential may be just as great. Natural adaptation to environment has been observed when populations of seed were increased in warmer or cooler climates (Smith, 1961; Zaleski, 1962). However, Simon et al. (1974), working with seed composites, found no s i g nificant yield differences as a result of multiplications in different regions, and the shifts they observed in growth-type means were regarded as minor. Yet adaptation is undoubtedly an important and continuous process, although its detailed effects are often by no means obvious. In Australia, for instance, the cultivar “Hunter River,” developed locally over many years, has consistently been shown to give greater long-term productivity than imported cultivars under many of the widely contrasting climates in which it is grown in this continent; yet it has not displayed any remarkable superiority in other countries. Experi-

218

K. R. CHRISTIAN

ments carried out by Chisci and Lessells (1960) support the hypothesis that varieties of local origin possess advantages which, other things being equal, result in the production of higher yields than those of outside varieties. Because of the great variability of plants within cultivars, it may even be unnecessary in many cases to seek unadapted genetic material outside the local region to obtain improved types. Mixtures of contrasting types are unlikely to be complementary, since the spaces previously occupied by plants that have died are taken by weeds rather than by the remaining alfalfa plants (Jackobs and Miller, 1973). The decline in stand density is for the most part an irreversible process, and cultural practices enabling self-seeding or interseeding would perhaps enhance production more than would any other single factor, not only by preventing stand deterioration but also by propagating genotypes most adapted to the environment. Various studies have illustrated the fact that genotypic variation is adequate for selection of specific characters. However, it has yet to be convincingly demonstrated that improvement in any one physiological character is not likely to be accompanied by compensatory changes that would negate any net effect on productivity. As examples, selection for higher phosphorus and lower calcium would probably at the same time increase potassium and reduce magnesium levels (HiU and Jung, 1975); fewer and thicker shoots might reduce shoot mortality, but could also result in more highly lignified herbage of lower digestibility; and, despite between-leaf relationships, SLW is poorly related to plant yield (Hart et al., 1972; Porter and Reynolds, 1975; Song and Walton, 1975). An integrated program of worldwide collection, recombination, and mild selection has been advocated by Hanson et al. (1972) to conserve and improve germplasm resources. On a more modest scale, many promising advances are already being made. Selective modification toward acclimatization has resulted in the development of a strain with exceptional tolerance to subarctic conditions (Klebesadel, 1971). T. J. Smith (1971) in a single cycle of natural selection under field conditions in a warm, humid region of Virginia obtained experimental strains from the interpollinated seed of “DU h i t s ” plants equal in persistence to those of the control variety “Williamsburg.” Hanson et al. (1972) used recurrent phenotypic selection for vigor and general appearance in developing multiple pest resistance. After three cycles of selection in the field, Frosheiser and Barnes (1973) obtained a 63% increase in resistance to Phytophthora megasperma, correlated with forage and root production in wet soils. Simpson (1974) has shown how the genetic potential to produce roots in low-calcium soils may be sought within cultivars by using clones propagated from cuttings which are grown in soils whose calcium availability gradually declines with depth.

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Although a better understanding of environmental effects is essential for the intelligent and economically viable upgrading of agricultural practices and techniques, our ability to modify the environment as a means of obtaining more favorable conditions for plant growth is limited and likely to become increasingly costly. On the other hand, our ability to modify plants to suit new environments, with the minimum expenditure of resources, may offer considerable opportunity.

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Scateni, W. J. 1972. Queensl. J. Agric. A n i m Sci 29,41-50. Schertz, D. L., and Miller, D. A. 1972. Agron. J. 64,660-664. Schmehl, W. R., Peech, M., and Bradfield, R. 1952. SoilSci 73, 11-21. Schonhorst, M. H., Davis, R. L., and Carter, A. S. 1957. Agron. J. 49, 142-143. Scott, T. W., and Erickson, A. E. 1964. Agron. J. 56,575-576. Seth, J., and Dexter, S. T. 1958. Agron. J. 50, 141-144. Shantz, H. L., and Piemeisel, L. N. 1927. J. Agric. Res. 34, 1093-1190. Sheridan, K. P., and McKee, G. W. 1968. Crop Sci 8,289-290. Silva, J. P. 1968. Diss. Abstr. 29, 1906B. Simon, U.,Kastenbauer, A., and Garrison, C. S . 1974. Crop Sci 14,682686. Simpson, J. R. 1965. Aust, J. Agric. Res. 16,915-926. Simpson, J. R. 1974. Proc. Int. Grassl. Congr., 12th 1, 330-7. Simpson, J. R., and Lipsett, J. 1973. Aust. J. Agric. Res. 24, 199-209. Singh, Y., and Winch, J. E. 1974. Can. J. Plant Sci 54,449. Smith, D. 1950.Agron. J. 42,398-401. Smith, D. 1951. Agron. J. 43,573-575. Smith, D. 1961. Can. J. Plant Sci 41,244-251. Smith, D. 1970a. Agric, Food Chem 18,652-656. Smith, D. 1970b. Agron. J. 62,520-523. Smith, D. 1971. Agron. J. 63,497-500. Smith, D., and Silva, J. P. 1969. Crop Sci 9,464467. Smith, D., and Struckmeyer, B. E. 1974. Crop Sci. 14,433436. Smith, D., Jacques, A. V. A., and Balasko, J. A. 1973. Crop Sci 13,553-556. Smith, T. J. 1971. Crop Sci 11, 27-28. Smoliak, S., Johnston, A., and Hanna, M. R. 1972. Can. J. Plant Sci 52,757-762. Snaydon, R. W. 1972a. Aust. J. Agric. Res. 23, 239-252. Snaydon, R. W. 1972b. Aust. J. Agric. Res. 23,253-256. Snaydon, R. W.1972c. Agric. Meteorof. 10,349-363. Song, S . P., and Walton, P. D. 1974. Crop Sci 14,663666. Song, S. P., and Walton, P. D. 1975. Crop Sci 15,649-652. Sonmor, L. G. 1963. Can. J. Soil Sci 43, 287-297. Sorensen, R. C., Penas, E. J., and Alexander, U. U. 1968. Agron. J. 60,20-23. Sprague, M. A., and Fuelleman, R. F. 1941. J. Am. Soc. Agron. 33,437447. Sprague, M. A., Hoover, M. M., Wright, M. J., MacDonald, H. A., and Brown, B. A. 1963. N.J., Agric. Exp. Stn., Bull. 804. Sprague, V. G., and Graber, L. F. 1938. J. Am . SOC.Agron. 30,986-997. Stanhill, G . 1962.Neth. J. Agric. Sci 10,247-253. Steinke,T. D. 1968.S. Afr. J. Agric. Sci 11,211-217. Sutton, C. D. 1969. J. Sci Food Agric. 20,l-3. Szeicz, G., Endrsdi, G., and Tajchman, S . 1969. Water Resour. Res. 5,380-394. Tadmor, N. H., Cohen, 0. P., Shanan, L., and Evenari, M. 1966. Proc. Int. Grassl. Congr., 10th pp. 897-906. Takasaki, Y. 1971. Proc, Crop Sci Soc. J p n 40,4&44. Takasaki, Y. 1972. Proc. Crop Sci Soc. Jpn. 41,205-212. Takasaki, Y., Takahashi, N., and Yokoyama, K. 1970. Proc. Crop Sci Soc. Jpn. 39, 144-150. Tanaka, T. N. 1971a. Proc. Crop Sci Soc. Jpn. 40,69-74. Tanaka, T. N. 1971b. Proc. Crop Sci Soc. Jpn. 40,306-310. Tanner, C. B., and Mamaril, C. P. 1969. Agron. J. 51,329-331. Taylor, H. M., and Gardner, H. R. 1963. SoilSci. 96, 153-156.

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Taylor, S . A., Haddock, J. L., and Pedersen, M. W. 1959. Agron. J. 51,357-360. Tewari, G. P., and Schmid, A. R. 1960. Agron. J. 52,267-269. Thomas, M. D., and Hill, G. R. 1949. In “Photosynthesis in Plants” (J. Franck and W. E. Loomis, eds.), pp. 19-52. Iowa State College Press, Ames, Iowa. Turrell, F. M. 1942. Am . J. B o t 29,400-415. Tysdal, H. M. 1946. J. Am . SOC.Agron. 38,515-535. Tysdal, H. M., and Kiesselbach, T. A. 1939. J. A m . Soc. Agron. 31, 513-519. Ueno, M., and Smith, D. 1970a. Agron. J. 62, 764-767. Ueno, M., and Smith, D. 1970b. Crop Sci. 10, 396-399. Ueno, M.,and Tsuchiya, S. 1968. J. Jpn. SOC.Grassl. Soc. 14, 188-192. Upchurch, R. P. 1951.Agron. J. 43,552-555. Upchurch, R. P., and Loworn, R. L. 1951. Agron. J. 43,493-498. Van Bavel, C. H. M. 1966. WaterResour. Res. 2 , 4 5 5 4 6 7 . Van Bavel, C. H. M. 1967. Agric. Meteorol. 4, 165-176. Van Riper, G. E. 1964. Agron. J. 56,45-50. Vartha, E. W. 1972. N.Z. J. Exp. Agric. 1, 29-34. Vartha, E. W., and Allison, R. M. 1973. N.Z. J. Exp. Agric. 1, 365-368. Vince-Prue, D. 1975. “Photoperiodism in Plants.” McGraw-Hill, New York. Vorhees, W. B., and Holt, R. F. 1969. Bull. Univ. Minn. Agric. Exp. Sin. 494. Vose, P. B., and Randall, P. J. 1962. Nature (London) 196, 85-86. Vough, L. R., and Marten, G. C. 1971. Agron. J. 6 3 , 4 0 4 2 . Wahab, H. A., and Chamblee, D. S. 1972. Agron. J. 64,713-716. Wallace, A. 1952. Agron. J. 4 4 , 5 7 4 0 . Walter, L. E., and Jensen, E. H. 1970. Crop Sci. 10,635438. Weir, W. C., Jones, L. G., and Meyer, J. H. 1960. J. Anim. Sci. 19,5-19. West, S . H., and Prine, G. M. 1960. Proc. Crop Sci. SOC.Fla. 20,93-98. Whitear, J. D., Hanley, F., and Ridgman, W. J. 1962. J. Agric. Sci. 59,415428. Whitehead, D. C. 1970. J. Br. Grassl. Soc. 25, 236-241. Willard, C. J. 1951. Adv. Agron. 3, 93-112. Williams, W. 1950. J. Br. Grassl. Soc. 5, 113-129. Willis, W. G., Stuteville, D. L., and Sorensen, E. L. 1969. Crop Sci. 9,637-640. Wilman, D. 1965. J. Agric. Sci, 65, 293-294. Wilsie, C. P., and Takahashi, M. 1937. J. Am. SOC.Agron. 29, 236-241. Wilson, D., and Cooper, J. P. 1969. New Phytol. 68,645455. Wolf, D. D., and Blaser, R. E. 1971a. Crop Sci. 11,55-58. Wolf, D. D., and Blaser, R. E. 1971b. Crop Sci. 11,479482. Wolf, D. D., and Blaser, R. E. 1972. Crop Sci. 12, 23-26. Wolf, D. D., Larson, K. L., and Smith, D. 1962. Crop Sci. 2,363-364. Yamada, T., and Suzuki, S. 1974. Proc. Znt. Grassl. Congr., 12th 3, 1016-1022. Yates, J. J., Russell, M. J., and Fergus, I. F. 1971. Aust. J. Exp. Anim. Husb. 11, 6 5 1 4 6 1 . Young, N. D., Fox, N. F., and Burns, M. A. 1958. Queensl. J. Agric. Sci. 16,199-215. Youngberg, H. W., Holt, D. A., and Lechtenberg, V. L. 1972. Agron. J. 64, 288-291. YouNs, M. A., Stickler, F. C., and Sorensen, E. L. 1963. Agron. J. 55, 177-1 82. Zaleski, A. 1954.J. Agric. Sci. 44,199-220. Zaleski, A. 1962. J. Br. Grassl. SOC.17,7-16.

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PHYSICAL PROPERTIES OF ALLOPHANE SOILS

’

T. Maeda. H . Takenaka. and B . P. W a r k e n t i d

I . Introduction .................................................. A . Allophane Soils .............................................. B. General Nature of Physical Properties of Allophane . . . . . . . . . . . . . . . . . . I1 . Index Properties ................................................ A. Grain Size Distribution ........................................ B . Plasticity ................................................... C. Surface Area and Heat of Wetting ................................ D. Mineral Density ............................................. E . Thermal Conductivity ......................................... 111. Structure of Allophane Soils ...................................... A . Description of Structure ....................................... B . Model for Physical Properties of AUophane ........................ IV. Physical Characteristics of Allophane Soils ........................... A . Volumechange ............................................. B . Water Retention ............................................. C. Water Transmission ....................... . . . . . . . . . . . . . . . . . . . . D Field Studies on Infdtration and Evaporation ....................... E . Water Available for Plant Use ................................... V. Soil Engineering ................................................ A . Compaction ................................................ B. Strength ................................................... C. Consolidation ............................................... D . Soil Stabilization ............................................ E . Adhesionandcohesion ....................................... References ....................................................

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I

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229 229 231 232 232 234 237 238 238 241 241 244 246 246 247 250 252 252 253 254 256 259 260 260 261

Introduction

A . ALLOPHANE SOILS

The term “allophane soils” used in the title is not uniquely defined. It is used here t o describe soils having observed properties common to soil materials which arise from weathering of pyroclastics . Volcanic ash soils is another term which Department of Agricultural Engineering. Hokkaido University. Sapporo. Japan .

’Department of Agricultural Engineering. University of Tokyo. Tokyo. Japan . Department of Renewable Resources. McGill University. Montreal. Canada .

229

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could have been used. The reader should interpret “allophane” in this less specific sense. The concept of allophane is changing as more becomes known about the material. Imogolite is now distinguished from allophane, but is included in our use of the term “allophane soils.” Imogolite, with its thread-shaped particles, has long-range order in one direction. It has a characteristic form in electron micrographs and has an identifiable X-ray diffraction pattern. Allophane, as the term is now used, has only short-range crystalline order, but does have an identifiable spherule form in electron micrographs. There are “amorphous materials” in soils which do not have this spherule form. It is likely that some of these materials are of pyroclastic origin. Possibly the term “andept” or “andosol” would have been a better choice to describe these soils. However, for the nonspecialist, the term “allophane soil” will probably best bring to mind the soils which we will be describing here. While these soils are dominated by allophanic properties, other minerals are often present in addition to allophane and imogolite. There are soils which contain small amounts of imogolite and/or allophane which would not be considered allophane soils here because they do not possess the physical properties associated with allophane. Allophane soils are widely distributed. They occur frequently in the Caribbean and Andean lands, as well as in the Pacific areas of Indonesia, Japan, New Zealand, and the United States. More studies on physical properties have been carried out in Japan than in any other country. One of the purposes of this review is to make the results of the published Japanese studies more readily available to readers of the English language. The references cited are mostly in Japanese, but usually have summaries in English. The more recent articles often have legends for figures and tables in English. Some of the papers are written in English. The justification for a review on physical properties of allophane soils is that they have distinctive properties which distinguish them from other soils. Soils can be divided into three groups on the basis of physical properties. In the first group, void characteristics determine physical properties, and void volume changes little with changes in water content. These soils, with sands as the example, can be treated as rigid, porous media. In the second group, the nature and extent of surfaces determines physical properties. Volume changes accompany water content changes; these changes are reversible even though they show hysteresis. Physical-chemical descriptions of behavior are often more useful than mechanical descriptions. Swelling clays are examples of soils in this group. In the allophane soils, of the third group, void characteristics rather than surface area determine physical properties. There are volume changes accompanying water content changes, but the effects are largely irreversible. The matrix changes on drying, and the dried soil can be considered a different material.

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

23 1

Much of this review will, therefore, be in the form of comparing physical properties of allophane soils with the properties measured for soils with crystalline clay minerals. It is assumed that most readers will be familiar with the latter.

B. GENERAL NATURE OF PHYSICAL PROPERTIES OF ALLOPHANE

The measured values of physical properties of allophane soils, in summary, show: they have low natural bulk density, high 15-bar water content, and high natural water content; that medium to low amounts of water are available to plants; they have high liquid limit and low plasticity index; they are difficult t o disperse; and that there are irreversible changes in all these properties on drying. Several good general summary descriptions of physical properties of allophane soils have been published. Swindale (1964) lists the following features:

. . .deep

soil profiles, usually with distinct depositional stratification, and normally friable in the upper part; topsoils as thick as one metre, and dark brown to black in color, containing humic compounds which are comparatively resistant to microbial decomposition; prominent yellowish brown to reddish brown subsoil colors with a smeary feel when the soil is wet; very light and porous profiles with a low bulk density and high water-holding capacity; rather weak structural aggregation, with easily destroyed porous peds lacking in cutans, and lack of horizontal differentiation in the subsoil except for the occurrence of duripans in some soils;. .. Smeary consistencies are marked only in soils in very humid or per-humid climates. The soils which form in per-humid climates tend to dry irreversibly when they are allowed to dry out in road cuts or banks. This feature of irreversible drying is a useful classification criteria, although the soils in the field never become dry enough to exhibit the property to any significant extent.

.

Fieldes and Claridge (1975) have summarized the early studies by Fieldes and his co-workers on New Zealand allophane soils:

. . . allophane in its early stages of formation could be visualized as gel-like fragments of random aluminosilicate held together by cross-linking at a relatively small number of sites. The fragments have an open internal structure, which originally, in the hydrogel state, enclosed much water. Until the water is removed, rearrangements of materials with more ordered structure cannot occur, and the moist clay has a weak “waxy” consistence. When the water is removed by drying, the structure collapses and further cross-linking takes place so that the process cannot be reversed; and the resultant material has considerable mechanical strength. Thus, it is not possible to reconstitute the moist hydrogel structure by rewetting, although the more compact xerogel is still open enough to have a strong affinity for water..

..

Allophane soils generally have a friable surface soil and massive structure in the subsoil, which however has a relatively high permeability. The friable structure of the surface soil is partly due t o effects of drying. Often allophane soils have several layers with very different physical properties which affect water movement and water available for plant use.

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Many properties, such as volume or water retention which decrease on drying, show an irreversible decrease beyond 10- to 15-bar suction. Physical properties of allophane soils do not show the dependence upon exchangeable cation which is prominent in soils with crystalline minerals. For example, Kubota (1971) showed that there was no difference in glycerol adsorbed on allophane with different exchangeable cations Mg, Ca, Sr, and Ba, and only about 4% lower adsorption with K as compared with Li. For bentonite there is a 20% difference for the divalent ions and nearly a 100%difference for the monovalent ions. Water vapor adsorption showed a similar pattern. I I. Index Properties

A. GRAIN SIZE DISTRIBUTION

The grain size distribution, also called particle size distribution or mechanical analysis, of a soil is the most widely used index property for physical properties of soils. Much effort has been spent in soil science on grain size measurements. For soils with crystalline clay minerals, especially in glaciated areas, and for clay contents less than 30%, one can predict many soil properties from the grain size distribution (Warkentin, 1972). However, for allophane soils the grain size is not an adequate index property. The index properties used for allophane soils include water retention (Flach, 1964; Colmet-Daage et al., 1967) and plasticity (Warkentin, 1972). Packard (1957) used surface area as a measure of clay content, as did Birrell (1966). Flach (1964) recommends using the 15-bar water retention to estimate clay content. The difficulty in obtaining dispersion, against both chemical and physical forces, and the uncertainty of what is the unit particle of an allophane soil, are the reasons for the limited usefulness of grain size in predicting physical properties of allophane soils. Chemical deflocculation is the problem with wet allophane subsoils, while in surface soils which have been dried the problem is cementing to form larger particles. These cementing bonds can be broken to different degrees. There is a considerable literature on the problems of dispersion of allophane soils (Gautheyrou et al., 1976). It is not possible from most of the studies to separate the effects of chemical deflocculation from physical dispersion. Therefore, the general term “dispersion” is used here. The difficulty of dispersing allophane soils has been noted by many people. Davies (1933) was one of the first to study the problem and recommended using 0.002 N HCl for dispersion. Kanno (1961) used 0.002 N HCl as a dispersant for Japanese allophane soils. Optimum pH for good dispersion of Kanto loam, both surface and subsoil, is in the pH range 2.5-3.5 (Tada and Yamazaki, 1963).

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

233

AUophane soils flocculate in sodium silicate solutions, but polyphosphates can sometimes be used as dispersants. Sodium pyrophosphate was a better dispersant than sodium metaphosphate for Indonesian soils (van Schuylenborgh, 1953). Low pH or calgon were used for Japanese soils (Kobo and Oba, 1964); the former works better for subsoils and the latter for surface soils with organic matter. Sherman et al. (1964) emphasized that the usual dispersing agents could not be used for allophane soils. Ultrasonic vibration was found to increase dispersion (Kobo and Oba, 1964), and is now generally used for allophane soils. Oba and Kobo (1965) found that ultrasonic dispersion released clay size grains from aggregates. A number of papers describe the use of this method (Gautheyrou et al., 1976). Espinoza et al. (1975) found that ultrasonic treatment still gave much lower values for clay content than did the estimate from the 15-bar water content, i.e., clay = FBP X 2.5. Ahmad and Prashad (1970) have taken a different approach t o dispersion of allophane soils. They found good dispersion by reversing the charge with zirconium to get a positively charged particle. Drying the sample decreases the measured clay content. This can be attributed to the cementing on drying. The phenomenon has been described by many workers, e.g., Sherman (1957), Birrell (1966), Wesley (1973). The magnitude of the effect varies with the particular allophane soil. Kubota (1972) measured the approximate soil suction at which irreversible bonding of clay into sand-size grains occurs on drying. The clay and silt contents began to decrease when the pF exceeded 3.5; fine sand-size grains were formed. Increases in coarse sand-size grains were not measured until about pF 5, at which time the clay and silt-size grains were at a minimum. The fine sand grains were then being bonded to coarse sand-size grains. No further changes in grain size distribution occurred at suctions above pF 5.5. Particles of pumice break down on stirring, and sand-size particles settle more slowly than expected because of internal pores (Youngberg and Dyrness, 1964). They also have a long wetting time because of entrapped air. Kobo (1 964) has summarized the Japanese studies on dispersion of allophane soils, and Colmet-Daage et al. (1972) report on an extensive series of tests of dispersion of allophane soils of the Antilles and Latin America. Their results are summarized as follows. There is no one best method which can be recommended. Surface and subsoils react differently, as do allophane soils containing different components such as gibbsite or halloysite. Both flocculation and incomplete physical dispersion occur. Undried samples always disperse more completely than air-dried or oven-dried samples, the difference being much larger for subsoils than for surface soils. Surface soil samples generally disperse better at high pH of 10 or 11 with ammonium or sodium hydroxide (the Kanto loam is an exception), while subsoils generally disperse better at pH 3 with HCl. Subsoils generally flocculate at high pH. Sodium pyrophosphate is an effective

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dispersant for surface soils, but metaphosphate is not. AUophane soils containing gibbsite are difficult to disperse in acid or basic suspensions. With small amounts of gibbsite, dispersion appears to be better at pH 3; with larger amounts the best dispersion is obtained at high pH. Soils with halloysite disperse at high pH, but not at low pH. Ultrasonic vibration is recommended for dispersion. Birrell and Fieldes (1952) had also noted that the presence of gibbsite makes allophane soils more difficult to disperse. Dispersion of surface soils at low pH might be attempted when solubilization or organic matter at high pH would interfere with subsequent measurements. The use of pyrophosphates might also interfere with other measurements on separated soil fractions. Control of pH is critical for dispersion at low pH, but not at high pH. The results given by Kubota (1972) are representative of the effect of different treatments on hydrandepts. On a B horizon sample the clay contents measured were as follows: 1Okc-300W sonic dispersion, 56%; standard shaking on moist sample, 31%; on air-dry sample, 5%; and on an oven-dry sample, 1%. Baba (1971) was able to disperse allophane soils in an alkaline medium only when ultrasonic treatment was used. Under these conditions sodium silicate was a more effective dispersant than sodium polymetaphosphate. While it is generally preferable to work with undried samples, this is not always possible. Undried samples may not be desirable if the soil contains predominantly sand and gravel because of the difficulty in obtaining a representative subsample of a wet soil. Grain size analysis, therefore, has a limited usefulness in characterizing allophane soils. The measurement should be done on field-moist samples (e.g., Schalscha et al., 1965) and the details of the method should be given. Since different allophanes react differently to dispersion treatments, some experimentation is necessary to obtain maximum dispersion (Colmet-Daage et al., 1972). The method most generally used for dispersion is ultrasonic vibration and low PH. B. PLASTICITY

The plasticity is one of the physical properties which distinguish allophane from crystalline materials. The name “allophane” comes from the striking change on drying of allophane clays. Glassy when wet, the allophanes become earthy on drying (Grim, 1953). The wet material is plastic, and the dry earthy material is nonplastic. Many papers have documented the plasticity limits, or Atterberg limits, and their change on drying (Birrell, 1951; Gradwell and Birrell, 1954; van Schuylenborgh, 1953; Yamazaki and Takenaka, 1965; Wesley, 1973; Warkentin and

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

235

Maeda, 1974; Kodani ef al., 1976; and others). Wet allophane soils have a high liquid limit, but also a high plastic limit, and hence a low range of water content over which they are plastic. As the samples are gradually dried, the liquid limit decreases more rapidly than the plastic limit. Highly allophanic soils become nonplastic before they reach the air-dry water content. The nonplastic state is where the plastic limit cannot be measured, or where its measured value equals or exceeds the liquid limit. Since the samples are completely rewetted during the determination of plasticity limits, the decreases in liquid and plastic limits on drying indicate an irreversible decrease in hydration of the allophane surfaces. The nature of these irreversible changes is discussed in Section 111, A. The values of plasticity limits are shown on a Cassagrande plot in Fig. 1. Crystalline clays have plasticity values which fall near the “A” line. The allophane samples fall far from the line, with the most allophanic samples having the highest liquid limit and the lowest plasticity index. This has suggested the use of plasticity values in classification of allophane soils (van Schuylenborgh, 1953; Gradwell and Birrell, 1954; Warkentin, 1972; Warkentin and Maeda, 1974). The measurement of plasticity is r e a d y made, while other measures of allophane are difficult to make. The intensity of allophanic characteristics would be highest for samples with high liquid limit and low plasticity index, and lowest for values approaching the A line. Samples which remain plastic on air-drying or oven-drying would have low allophanic characteristics. The difficulty in using plasticity values for classification is that the measured values depend upon degree of previous drying and upon content of organic matter. Since the drying history of surface soil samples is usually not known, air-dry samples may have to be used. The method might be more suitable for subsoils.

120

c

Liouid Limit

FIG. 1. Cassagrande plasticity chart with values for Japanese allophane soils (from Yamazaki and Takenaka, 1965). 0, Fresh soil; 0 , air-dried soil.

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Organic matter contributes t o the water held at the liquid limit (Kodani et aZ., 1976), an effect which decreases irreversibly on drying. Maeda et al. (1976) have shown the separate contributions of organic matter and mineral matter to the liquid limit of allophane soils (Table I). The liquid limit is increased from 1.5 t o 3% for each 1% organic matter in the samples they used. Their results indicate that not all the organic matter in a soil sample contributed to the liquid limit. Bonfils and Moinereau (1971) found that both liquid limit (L.L.) and plastic limit (P.L.) were strongly related to organic matter content (O.M.); the equations were: L.L. = 2.7 O.M. + 41 and P.L. = 2.7 O.M. + 34. The plasticity index was not correlated with organic content. The activity values (ratio of plasticity index to clay content) measured for allophanes are variable. Northey (1966) gives values of 1.2 t o 1.5, Wesley (1973) gives value below 0.6. The meaning of these values is uncertain because the measurement of clay content is difficult. The measurement of plasticity is more difficult for allophane samples than for crystalline clays, and the precision is lower. This results from the low range of water content over which the samples are plastic. The slope of the liquid limit determination (water content versus log number of blows) is also more variable for allophane clays than for crystalline clays. The one-point liquid limit method (Sowers, 1965) cannot be recommended for allophanes. The degree of remolding and working of the soil with water affects the liquid limit, especially of subsoils (Ikegami and Tachiiri, 1966). Measurements of liquid limit by Soma and Maeda (1974) on soils which were gradually dried show a sharp break at a specific water content where irreversible changes occur (Fig. 2). Drying does not produce irreversible change for the soil shown until the water content falls below 100%. The shrinkage limit also occurs at this water content. TABLE I Effect of Organic Matter on Liquid Limit of Allophane Soil' Water content at liquid limit Organic Soil content (%) A

29

B

20 20 17 23 11

C

D E F

Natural water content (%) 142 116 119 133 108 44

'From Maeda etul. (1976).

Decrease in liquid limit on air-drying

Whole Organic matter Due to organic Due to soil (%) removed (%) matter (%) allophane (%) 180 164 147

151 172 62

83 72 72 107 107 42

33 28 36 8 52 2

36 21 15 40 5 3

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

237

180

.=E 160 ._

-I 140 v .S

.F 120 -I

Q

.-

,, 1.4-

I00 I

*.-*

2b

40

60 80 IiO IhO I40 I60 Initial Water Content

FIG. 2. Decrease of liquid limit and volume of allophane soil o n gradual drying (from Soma and Maeda, 1974).

Freeze-drying produces less particle bonding than air-drying, and results in higher measured plasticity (Warkentin and Maeda, 1974). Ultrasonic vibration increases the measured liquid limit (Soma and Maeda, 1974) because it causes some dispersion. There is little change in liquid limit with different exchangeable cations. For some samples the Na-soil has a slightly higher liquid limit than the Ca-soil, for others it is reversed (Yazawa, 1976). The flow index (slope of the water content versus number of blows curve in the determination of liquid limit) is also not consistently different for Na or Ca allophane soils (Yazawa, 1976).

C. SURFACE AREA AND HEAT O F WETTING

Measurements of surface area for allophane soils have been reviewed recently by Wada and Harward (1974). This literature will not be reviewed in detail here. The surface area values are high, in the range of 300-600 mZg-’, but some of this surface is not accessible to large molecules. This is not equivalent t o the internal and external surfaces of swelling clays, but is due to small size of voids and small necks leading to voids. The heats of adsorption indicate that physical adsorption rather than chemical adsorption is dominant (Fieldes and Claridge, 1975). Total surface area is often calculated from the amount of ethylene glycol adsorbed, and external surface is measured from nitrogen adsorption; internal surface area is estimated from the difference. Aomine and Egashira (1970) found ratios of total to internal surface from 2.3 t o 3.0, while Fieldes and Claridge (1975) report ratios of 1.8 to 2.6 when the nitrogen surface was measured after heating to 600°C. Egashira and Aomine (1974) found that total surface area measured on samples vacuum-dried over P2 O5 was higher than for oven-dried samples.

23 8

T.MAEDA ET AL.

Aomine and Egashira (1970) measured the heat of immersion of allophane soils in comparison with soils containing crystalline minerals. They found that for equal surface areas, allophane soils had heats of immersion about twice as large as montmorillonite soils. The ratio of heat of immersion to surface area for allophane and from 0.023 to 0.029 for ranged from 0.047 to 0.056 cal montmorillonite soils. The exchangeable cation had only a small effect on heat of immersion of allophane soils. For montmorillonite soils, hydration of exchangeable cations is a more important part of total heat of immersion. Water molecules are bonded more strongly on allophane surfaces than on montmorillonite surfaces. When the heat of immersion was plotted against initial water content of the allophane soils, the oven-dry samples fell below the smooth curve joining the vacuum-dried and moist samples. This indicated to the authors that oven drying had altered the nature of the allophane surface. Maeda er al. (1976) measured heat of wetting values for an allophane soil ranging from 7.3 cal g-' at 4% organic matter to 11.2 cal g-' at 26% organic matter. D. MINERAL DENSITY

Some measured values of mineral density, or specific gravity, of wet allophanes are low, in the range of 1.8-1.9 g cm-3 (Fieldes and Claridge, 1975). These values had been accepted in earlier studies. However, other measurements show values of 2.7 or higher. Forsythe etal. (1964) quote values of 2.7-2.9 g ~ m - ~ . Wada and Wada (1975) measured values of 2.72 to 2.78. They took special precautions to remove entrapped air from the samples. If the unit particle of allophane is accepted to be a hollow spherule of 50 A outside diameter and about 30 A inside diameter, water movement into and out of this sphere would be difficult and could account for the low mineral density sometimes measured. Bonfils and Moinereau (1971) measured values of 2.32 to 2.70, the lower values being for horizons with large amounts of organic matter-about 25%. E. THERMAL CONDUCTIVITY

The thermal conductivity of a soil depends upon the conductivities of the components-mineral, organic, water, and air. Because the path of heat transfer from one component to another cannot be easily specified, the conductivity of a soil cannot be readily calculated from the conductivities of the Components. Models exist for some heat flow paths, but in general the preceding statement is true. However, soil thermal conductivities vary in a predictable way with properties of the components. On this basis allophane soils would be expected t o

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

239

have thermal conductivity and thermal diffusivity values which are lower than the corresponding values for soils with crystalline clay minerals. Diffusivity is the ratio of conductivity to heat capacity and is the constant which relates temperature changes in the soil to the temperature gradient. The conductivity of glass is lower than that of clay minerals or quartz (Cochran et al., 1967). Allophane soils have a lower bulk density than soils with crystalline minerals; this should result in lower thermal conductivity. The higher water content would give a higher heat capacity and hence lower thermal diffusivity. Some of the results obtained are summarized in Table 11. Yakuwa (1 943) made extensive measurements on soil temperature and thermal properties of different soils in Japan, including allophane soils. Higashi (1951) measured a value of 0.32 cal g-' O C - ' for specific heat of dry allophane soil. Thermal diffusivities were calculated from amplitude ratios and phase differences of temperature waves; the phase differences gave more consistent results. While the dense packing seems to give higher values of diffusivity, the scatter was such that no conclusions could be drawn on effect of bulk density (Table 11). The maximum in the diffusivity curve occurred at 50% water on a weight basis or about 0.25 on a volumetric basis. Thermal conductivity, calculated from specific heat and diffusivity, was higher at high bulk density. ~ ; dense packing The loose packing had a bulk density of around 0.5 g ~ m - the varied from 1.0 at 0% water to 0.7 g cm-3 at 50% water. Higashi (1952) also measured thermal properties of frozen allophane soils. The thermal diffusivity is similar for frozen and unfrozen soils below a water content of about 30%, but at higher water contents the diffusivity of frozen soils increases very quickly. Cochran et al. (1967), using a line heat source probe, found that thermal conductivity of a pumice material was very low, only 'slightly higher than for a peat soil (Table 11). They used this to explain low night temperature and the high incidence of frost in pumice soil areas of Oregon. The maximum in the diffusivity-water content curve occurred at a low volumetric water content of 0.04 for the C horizons of the soil. Maeda (1968) measured temperature gradients in soils near Sapporo, Japan. He calculated thermal diffusivities from amplitude ratios and phase differences. The diffusivity of fine pumice was higher than for an alluvial soil, and the allophane soil was the lowest (Table 11). Kasubuchi (1975a) found that the specific heat of allophane clay (0.229 cal g-' "C-') was higher than bentonite (0.209), kaolin (0.201), or quartz (0.170). Kasubuchi (1 975b) measured thermal conductivity of different soils using a line heat source probe. The values again show conductivity and diffusivity values for allophane which are 20 to 35% of those for soils with crystalline minerals. The higher heat capacity and lower heat conductivity of allophane soils results in slower temperature changes.

TABLE I1 Measured Thermal Properties of Allophane Soils

Material

Bulk density (g cm--' )

Water content (w, %)

1.15 Loose Loose Dense Dense

11 0 50 0

Heat Thermal capacity conductivity (cal ~ r n - ~ ~ c - ' )(mcal cm-' sec-' "C' ) ~

Pumice soil Allophane soil

Crown glass Pumice

0 40% 0 40

0.15 0.55 0.30 0.70

-

0.76

Clay soil Allophane soil Alluvial soil Fine pumice Allophane Alluvial soil Diliivial soil Black andosol Brown andosol

50

0.32 0.20 0.39 0.29 0.53

0.2-0.3 0.5 0.5 0.77 0.72

40 40 40 40 40

Reference

~~

1.1 0.22 0.81 0.32 1.23 2.3 0.37 I .25 0.60 3.80 -

-

Thermal diffusivity [cm2sec-' (X

0.5-0.7 2.0-2.5 3-4

0.6 0.8

3.4 1.13 -

2.34 -

2.6 2. I 2.4 3.5 6.6 1.5-1.7 3.74.0 4.5-5.5 1.5 1.7

Yakuwa (1943) Higashi (1951) Higashi (1951) Higashi (1951) Higashi (1951) Cochran et al. (1967) Cochran ef a!. (1967) Cochran et nl. (1967) Cochran et al. (1967) Cochran et al. (1967) Maeda (196 8) Maeda (1968) Maeda (1968) Kasubuchi (1975b) Kasubuchi (1975b) Kasubuchi (1 975b) Miyazawa and Konno (1976) Miyazawa and Konno (1976)

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

24 1

The values reported by Miyazawa and Konno (1976) confirm the other measurements (Table 11). They found a maximum thermal diffusivity of 1.8 t o 2.0 cm2 sec-’ at a volumetric water content of 0.50. They used these values t o describe the thermal regime of the soils. Ill. Structure of Allophane Soils

A. DESCRIPTION OF STRUCTURE

A major difficulty in describing physical properties of allophane soils is a lack of understanding of the structure (size, shape, and arrangement of particles and voids) at different levels of observation. This is compounded by the apparent change in nature as well as amount of surface exposed as the soil dries. These changes are irreversible when the sample is dried at high suction, although they are usually reversible at low suction. The structure at the lowest level, 10-100 A, is being defined in recent studies, and structure at the highest level, 1-10 mm, is also known. But little information exists at intermediate levels, for example over the range of void sizes retaining water for plant use. The differences in physical properties among allophane soils cannot, as yet, be related to differences in structure; often the differing physical properties are attributed to different amounts of allophane present. This is reminiscent of the situation 70 years ago when different soil properties were explained by different contents of “kaolin” in the soil. An exception to this lack of understanding is pumice, where the physical properties can be related to size, shape, continuity, and strength of voids (Sasaki et al., 1969). The terminology used to describe structure in the 0.01 to 10 pm range varies with the discipline, or even with the author. No attempt will be made in this review to suggest a preferred terminology. Structure will be used as defined by Brewer (1964) to include size, shape, and arrangement of particles and voids; fabric is the component of structure which describes arrangement. The structure of allophanes in the 1 - to 10-mm range is given in many field descriptions. The total porosity is high, with typical bulk density values from 0.3 to 0.8 g ~ m - Some ~ . of the low-silica allophane soils in Hawaii have bulk density values around 0.1 g cm-3 (Sherman et al., 1964). There is a marked difference between surface soil and subsoil, due mostly to effects of drying. The topsoil usually shows good aggregation, with well-defined and stable interaggregate voids. The subsoils show the properties of undried allophane, with restricted profile development. The porosity is high and tubular pores are present, although these pores have restrictions that limit percolation of water (Tabuchi et al., 1963).

242

T. MAEDA ET AL.

The structure of imogolite in the 10- to 100-A range is known from electron micrographs supplemented by X-ray diffraction, electron diffraction, and salt absorption measurements (Wada et aZ., 1970; Wada and Henmi, 1972). The unit is a hollow tube with inside and outside diameters of 7-10 and 17-21 A, respectively. A number of tubes lying parallel form threads of 100 to 300 A in diameter. Three different classes of pores would be present in such a material: intraunit pores of about 10 A in diameter, intrathread (interunit) pores of the same or slightly smaller diameter, and interthread pores of several hundred Angstrom (A) units diameter. The “unit particle” of allophane has been defined (Kitagawa, 1971) as a spherical particle with a diameter of about 55 A (Birrell and Fieldes, 1952). The density of this particle is about 1.9 g ~ m - Gtagawa ~. (1971) has assumed that these unit particle spheres exist in close packing in the air-dry state. This forms the “microaggregates” commonly found. On heating, the layer of adsorbed water is lost, and the spheres come closer together. Grinding distorts the unit particle spheres allowing closer packing, which, in the electron micrographs, gives the appearance of sheets rather than the individual spherical particles of the dried sample. The close packing of these 55-A diameter units would again result in voids about 10 A in diameter, so the water absorption properties would be simdar t o imogolite. Irregular packing would produce some larger pores. These models, therefore, explain many observed properties. The decrease in surface area on grinding is due to collapse of some of the small pores. Kitagawa (1971) has shown that calculation of particle size from surface area and density also gives values of about 55 A diameter. He found that phosphate adsorption was not decreased by grinding on drying, which argues against any chemical change in the surface, specifically in the number of hydroxyls at the surface. Interparticle bonding is then by physical forces that would be weaker than if chemical bonding were involved. These measurements were made on a series of allophane soils from Japan. It is not known whether all allophanes have this same “unit particle.” Measurements by Rousseaux and Warkentin (1976) of water vapor absorption show a maximum in pore volume at diameters of 7-10 A for allophanes from the Caribbean and from Japan. The Caribbean samples show a narrower size distribution that could result from closer and more regular packing of unit particles. Fujiwara and Baba (1973) calculated effective pore size from nitrogen absorption measurements, and found a maximum at 25 A. The volume of these 25-A pores was about one-twentieth of the volume of 10-A pores found from water absorption by Rousseaux and Warkentin (1976). These measurements confirm the unit particle in allophane soils. Measurements of water vapor absorption are of necessity made on dried samples. It is reasonable to assume that undried samples consist of the same unit

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

243

particles, but that they are further apart and more irregular. Such a model would explain water retention, plasticity, etc. It is more difficult to relate differences in these properties among soils, wet or dry, to different structures in the model. Some allophane soils become nonplastic on air drying, others do not. This could be due to differences in arrangement of unit particles, or to different nonallophane components in the soils. Also, it is not known how differences in chemical composition such as the AI/Si ratio affect the structure, although these differences are correlated to differences in physical properties (Rousseaux and Warkentin, 1976). The Caribbean samples, for example, have lower Si02/A1203ratios than the Japanese samples. Ito (1 964) postulated that allophane structure consisted of individual particles and massive particles (structure units), with only the latter affected by drying. The individual particles retain their undried properties, and different phases of shrinkage are due to different effects of individual particles and structure units. The irreversible changes in physical properties of allophane soils on drying set them apart as a separate group of soils. In the extreme, allophane soils change from highly plastic when wet, to sandy when dry. This has been described by many workers, e.g., Sherman et al. (1964), who suggest research on changing the colloidal properties of allophane soils by promoting dehydration. The particles become cemented together to form units of sand size that are sufficiently strong to withstand the usual manipulation in field or laboratory. Neither the forces involved in this cementing nor the fabric of the units have been adequately described. A tentative model for structure is described in Section 111, B. The observed pore structure of allophane soils has been described by a number of workers in relation to permeability. Tabuchi et al. (1963) described the channels for water flow that they observed in thin sections of surface and subsoils. Interaggregate pores conducted water in surface soils, tubelike pores were found to conduct water in subsoils. Takenaka et al. (1963) report similar results. Nagata (1963) found that plots of log air permeability against air porosity were linear functions which could be related to the kind of structure in the soil sample. For some soils log K, versus va consisted of two straight-line portions, depending upon changes in structure with void size. A more detailed description is available for the pores in pumice (Borchardt et al., 1968; Maeda et al., 1970; Tsujinaka et aZ., 1970). On the basis of waterretention measurements carried out in different ways, Maeda et al. (1970) distinguished dead, active, semiactive, secondary active, and semidead pores. The active and semiactive pores usually occupy the largest volume, but semidead and dead pore space may be large in some samples of pumice. The pumice materials have been described by Sasalu (1 957). Some information on structure, especially on the fabric component, can be obtained from measurements of rheotropy or thixotropy of allophane soils. The behavior of allophane on mechanical manipuiation is usually not true thixo-

244

T. MAEDA ET AL.

tropy, i.e., a reversible sol/gel transformation. The changes on remolding described by Takenaka and Yasutomi (1965) are discussed in Section V, B. A number of papers have reported experiments with soil conditioning chemicals; the general impression is that these chemicals are only marginally effective in promoting aggregation in allophane soils. This may be partly due t o mixing the chemicals with soils at water contents which are not optimum for aggregation. Sudo and Suzuki (1963) found that sodium alginate increased stable aggregates in an allophane soil but the polyelectrolyte CMC did not. They report, in their literature review, that additives are generally considered to have little effect on aggregation of allophane soils. Kawaguchi et al. (1963) report that bentonite added to polyvinyl alcohol is effective in aggregation. Fujioka et d. (1965) found an effect of soil conditioners on allophane soils, but Terasawa (1 967) found very little effect of synthetic polymers on aggregate formation.

B. MODEL FOR PHYSICAL PROPERTIES O F ALLOPHANE

What is required is a model for structure, including fabric, of allophane soils. This model must explain the known physical properties of allophane and their changes on drying. The model should predict other physical properties. It should also be related t o important differences in chemical composition and properties, for example the Si/Al ratio. The model must describe structure in the 0.01- to 100-pm range, where physical properties can be explained. Measurements to date on allophane soils allow only a general specification of this model. The unit particle of about 50 A is well established and can be taken as the starting point. This particle has an “internal” water content. The unit particle has been established in dried samples and is assumed to be present also in wet samples. These unit particles are then weakly bonded together to form domains in the diameter range of 0.01 to 1 pm. The modal size may be 0.05 to 0.1 pm. The voids between particles in these domains account for the large water retention above 15-bar suction. Drying brings the particles closer together, increasing the bulk density of the domains and decreasing water retention above 15 bar. Irreversible changes occur when the unit particles come sufficiently close together to allow strong bonding between unit particles. These domain units may be the clay-size grains measured in a grain size determination. They are not broken up on remolding and account for the high water content at the plastic limit. They are the units that move on plastic readjustment. Organic molecules are held within the domains. The domains are arranged in clusters in the size range of 1-100 pm. These units have weaker bonding than the domains. Remolding breaks up the clusters, releasing water held within the cluster. Drying causes shrinkage of the domains and rearrangement into clusters of highei bulk density. In some allophane soils

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

245

the bonds holding the clusters together after drying are strong, and the clusters become the units of dried soils. Bonding within clusters would then aIso contribute to irreversibility. This may be true only in soils that are composed entirely of allophane and do not have crystalline minerals or oxides mixed in. Water in the plant-available range is held withm the clusters. The water-retention curves for allophane soils show an approximately linear change of water content between 0.03 and 1 bar, with decreasing amounts of water retained below and above these values. The break at 1 bar indicates a change at void diameters of 30 pm; the upper boundary of cluster size could be drawn there. This would put much of the plant available water between clusters. Kubota (1971) has studied the formation of units in the sand- and silt-size range. Drying is necessary for pedogenic formation of these units. These units fall within the range defined here as “clusters.” These units are stable against dispersion if the allophane soil has been dried. Dehydration alone can cause irreversible binding. The formation of these clusters is best studied on subsoil samples of allophane soils that have been continually moist. Presumably the clusters are already present in surface soil horizons that have been previously dried. Kubota (1971) divides the clay-size grains into three types on the basis of their potential for forming sand- and silt-size grains on drying. The clay content was measured on sonic-dispersed samples of subsoils of wet allophanes. Type I is active free clay which can form large grains on air drying. This is measured as the difference between clay content of moist and air-dry soils on shaking. Type I1 is the aggregated clay that is released from moist soils by sonic dispersion. These are pedogenically formed aggregates. Type 111 is inactive free clay not affected by air-drying. Ttus is the measured clay content of an air-dry soil. Type I clay content increases from the Ap to the B2 horizon, while Type 111 decreases. Type I1 remains approximately constant with depth. Monolayer adsorption of water vapor on allophane is strong but multilayer adsorption of more than two water layers is weak compared with layer silicate minerals. Therefore unit particles of allophane can come in close contact. Kubota (1971) also states that hydroxyaluminum groups are the site for adsorption of water molecules; they are also the sites for bonding of unit particles in an irreversible aggregation. The details remain to be fdled into this model. The size ranges of the fabric units, domains, and clusters, have been chosen mostly for convenience of description. They will undoubtedly need refinement. Neither the size boundaries nor the terminology of domains and clusters are suggested as being definitive. They are both used here for convenience. The clusters may be better described as microaggregates. However, Kitagawa (1971) refers to the unit particles of 55 A as microaggregates, and Kubota (1971) refers to sand- and silt-size units as aggregates.

246

T.MAEDA ET AL. IV. Physical Characteristics of Allophane Soils

A. VOLUME CHANGE

Wet allophane soils show a large volume decrease on air-drying, and a limited volume increase on rewetting. Most of the volume change is irreversible. This distinguishes allophane soils from swelling mineral soils, where the volume changes are more nearly reversible. The amount of shrinkage of an allophane soil depends upon the initial water content and the fabric changes during drying, both of which depend upon the allophane content. Many physical properties change in association with this volume decrease, e.g., permeability increases. However, volume change in the field cannot be predicted quantitatively from shrinkage measured on small samples because the boundary conditions are different. Wet allophane samples dried to intermediate water contents, suctions below 10-20 bars, will regain most of the volume on rewetting. Volume change is approximately reversible over this range (Takenaka, 1961). Volume change curves can be plotted as changes in measured volume, linear dimensions, bulk density, or void ratio with change in water content. The measurements are not precise because it is difficult to measure volume repeatedly on a sample as the water content changes. The precision is sufficient t o characterize shrinkage on drying, but greater precision is desirable in describing volume change for the water-retention curve (Section IV, B). The general nature of the shrinkage curves is shown in Fig. 3 for a highly allophanic soil (Cl) from Dominica, West Indies, and for a soil (Nl) from Hokkaido, Japan, with low allophanic properties (Warkentin and Maeda, 1974). There is a break in the volume change curve which can be called a shrinkage limit, but the volume change at higher water contents is not “normal” shrinkage where volume decrease equals water content decrease (Takenaka, 1961; Ito, 1964). The shrinkage limit is less pronounced and occurs at a higher water content as allophane content increases. The shrinkage limit occurs between 50 and 100% water for allophane soils, a much higher value than for crystalline clays (Takenaka, 1965; Warkentin and Maeda, 1974; Soma and Maeda, 1974). Takenaka (1965) found that the shrinkage limit occurred near pF 6 for a sample of Kanto loam. He related shrinkage to soil suction values and showed that the amount of shrinkage depended upon the rate of drying. The measured water content at the shrinkage limit also increases with increasing organic matter content of the soil (Takenaka, 1973). He measured a 15% increase in shrinkage limit for 10% increase in organic matter up to 25%. Remolding does not change the value of the shrinkage limit (Takenaka, 1965), but drying increases the shrinkage limit. The usefulness of the shrinkage limit is in the information it gives about fabric and structure of allophane soils. The high water content at which the slope of

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

0

20

40

60

80

100

I20

247

140

Water Conieni, %

FIG. 3. Shrinkage curves for two allophane soils. Reproduced from Soil Science Society of America Proceedings, Volume 38, Page 375, 1974 by permission of the Soil Science Society of America. X , Field moisture; @, air dry; 0,oven dry.

the shrinkage curve changes and the high value of residual shrinkage (between the shrinkage limit and zero water content) indicates a random arrangement of units. Measured shrinkage is isotropic, again indicating random arrangement. For crystalline minerals, the shrinkage limit is lowest for high swelling soils. The shrinkage limit is, therefore, diagnostic for allophane soils (Warkentin and Maeda, 1974). Despite the large amount of shrinkage on drying, one would predict little visible cracking for allophane soils in the field because the shrinkage is taken up in small spaces between clusters (Section 111, B). Cohesion of allaphane is low and it decreases if the samples are dried (Soma and Maeda, 1974). AUophane soils do not form dry clods with dimensions of tenths of a meter. The effect of remolding a soil depends upon its degree of consolidation (Croney and Coleman, 1954). Remolding an overconsolidated soil exposes new surfaces for water retention. AUophane soils are underconsolidated. Remolding breaks some of the fabric bonds, decreases the soil suction, and increases the amount of shrinkage (Takenaka, 1965).

B. WATER RETENTION

This section will deal with soil water characteristics measured on allophane soil samples in the laboratory; the mechanisms of water retention will be discussed. The field water regime is described in Section IV, D.

248

T. MAEDA ET AL.

It has been difficult to relate soil suction measured in the laboratory to the water regime of allophane soils in the field. Only two variables-water content and suction-are usually measured; while the complete description of the waterretention curve of an allophane soil requires the specification of four variableswater content, suction, sample volume or percent water saturation, and initial degree on drying of the sample. Only for coarse-grained soils, where suction and water content determine the system, have water-retention curves been useful in predicting the field water regime. Measurements in the laboratory on finegrained soils, especially swelling clays, have not been useful in predicting soil water behavior in the field. The water-retention curve for swelling crystalline clay soils requires specification of three variables-water content, suction, and volume. If the structure is disturbed on sampling this can be considered a fourth variable. Initial bulk density is assumed to be the same as that in the field, even though this is not always assured. Another difficulty in applying laboratory measurements to the field is the unreliability of water-retention measurements during wetting of fine-grained soils. The measurements are almost always made only on the drying part of the cycle. Therefore, the main use for water-retention measurements for allophane soils is in characterizing soil surfaces or void-size properties. In addition, ColmetDaage and his colleagues (e.g., Colmet-Daage et al., 1967) have used p F values for wet and dry allophane soils as an index property (see Section 11, A). Hughes and Foster (1970) have suggested that the water content at a specific suction can be used to rank the degree of disorder, or the amount of extractable materials, in halloysite and allophane. Galindo-Griffith (1974) showed that the 15-bar percentage was not correlated with surface area, but was correlated with reactive alumina and with the point of zero charge. Many physical properties of allophane soils are interrelated, as has been shown in a number of papers (e.g., Warkentin and Maeda, 1974). Water retention by allophane soils is determined by the size distribution of voids, not by the amount of surface area (e.g., Fujiwara and Baba, 1973). This fact differentiates allophane soils from soils with swelling clay minerals, The amount of water held by wet allophane samples is lllgh (Table 111); the very high water contents at 15-bar suction are striking. This results from the large volume of small voids. Drying the samples decreases water retention at any suction value. These measurements have been made by a number of people. Msono et ul. (1953) made a detailed study of water-retention characteristics of a number of Japanese allophane soils. Colmet-Daage and his co-workers (1967, 1970) have published measurements for a wide range of allophane soils from the Caribbean, Central America, and South America. These reports provide very valuable source material on properties of allophane soils, A complete list of papers is available in the bibliography by Gautheyrou et al. (1976).

249

PHYSICAL PROPERTIES OF ALLOPHANE SOILS TABLE 111 Measured Water Content at Different Suctions for Allophane Soils Water content at suction Sample Kuriyagawa B1-Wet -Air-dried Dominica -Wet

0.001 bar 0.3 or 0.5 bar 15 bar

Reference

275 170

160 80

80 50

Misono ef QI. (1953) Misono et QI. (1953)

145

90

70

130

70

50

Maeda and Warkentin (1975) Maeda and Warkentin (1975)

-

138 235

100 174

-

207

83

-Dry Ecuador -Fresh

-

143

42

-

255

192

-Dry Ecuador 0-1 10 cm, weakly allophanic -Fresh

-

36

33

-

65

45

-

40

30

-

150

115

-

40

35

-Air-dried Hawaii Hydrandep t -Apl -B24 Martinique -Fresh

-Dry 50-300 cm, strongly allophanic -Fresh -Dry

Flach (1964)

Colmet-Daage er QI. (1972) Colmet-Daage et al. (1972) Colmet-Daage et aL (1967) Colmet-Daage et al. (1967) Colmet-Daage et al. (1967) Colmet-Daage er at. (1967) Colmet-Daage er al. (1967) Colmet-Daage et QI. (1967)

The measurements by Colmet-Daage and Cucalon (1965) illustrate the large decreases on drying in water content at p F 4.2 and 2.8. The amount of water held between these two suction values, an estimate of plant-available water, also decreases on drying. In extreme cases for certain horizons this decrease was from 45% to 3% and from 68% to 5% by weight. Soils with a high content of allophane have an S-shaped water retention curve, similar t o the shape for a coarse-grained soil. The approximately linear portion on the water-content-log-suction plot is between 0.01 and 1-bar suction (Maeda and Warkentin, 1975). For swelling crystalline clays this linear portion extends from less than 0.01 bar to around 100 bar.

250

T. MAEDA ET AL.

Forsythe (1972) states that the loss in water retention on drying is greater at lower suctions than at high suctions. Maeda and Warkentin (1975) confirmed this for samples with moderate allophanic properties; for highly allophanic soils the effect appeared to be reversed. Forsythe (1972) found that volumetric water content increased on air-drying; Maeda and Warkentin (1975) found a lower volumetric water content and lower percent saturation of samples after drying. Parfitt and Scotter (1972) report on an allophane soil from Papua, New Guinea, with a low 15-bar percentage of 36% and a 0.1-bar percentage of 120%. This results in a high content of plant-available water. Colmet-Daage et al. (1967) checked the influence of organic matter on water retention by allophanic soils. They realized that treatment with peroxide could aid in dispersion, so that the measured difference could not be attributed solely to organic matter. They found that treatment with hydrogen peroxide to remove organic matter had no influence on water retention at pF 2.5. At p F 4.2 some of the treated samples had lower and some had higher water retention than the control samples. They concluded that organic matter had only a small influence on water retention for the soils which they studied. However, in humic dlophane soils, the organic matter content is important in water retention. Swindale (1964) reports that for some Hawaiian soils, percent field water content (w) increases with percent organic matter (O.M.): w = 4.2 + 34.7 O.M. Takenaka (1973) and Maeda et al. (1976) pointed out that water retention by allophane soils high in organic matter content was especially decreased on drying. A number of authors have divided the water-retention curve into three or more classes of water (e.g., Misono et aL, 1953). The breaks in the water-retention curves indicate some validity in this approach, but there is no evidence that forces of water retention are different and can be separated in this way. The retention appears to be due to voids of different sizes except at high suction values, larger than 100 bar, where surface adsorption is involved. The waterretention curve at suctions below 100 bar can then be used to obtain a qualitative determination of void size distribution (Misono et al., 1953; Maeda and Warkentin, 1975). Masujima (1962) found that different exchangeable cations did not change the water retained between 10 and 100 bar.

C. WATER TRANSMISSION

Rate of water transmission through allophane soils is high, due t o the low bulk density and the granular structure of surface horizons (Table IV). At the same void ratio, allophane soils have a higher saturated hydraulic conductivity than soils with montmorillonite (Maeda and Warkentin, 1975). Drying increases the saturated hydraulic conductivity at constant bulk density. The increase is at least two orders of magnitude for highly allophanic soils but

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

25 1

TABLE IV Saturated Hydraulic Conductivity of Allophane Soils

Sample Onuma -Wet Dominica -Wet -Air-dry Ecuador -Dry Costa Rica -Field Kanto loam -Surface -Subsoil Averages

Db (g ~ m1 - ~

1.0 1.0

Saturated K (cm sec-' )

Reference

6 X lo4

Kubota (1972)

3x 1 x 10-5

Maeda and Warkentin (1975) Maeda and Warkentin (1975)

2 x 10-~

Colmet-Daage el al. (1967)

3 x 10-~

Forsythe (1975)

10-2

Tabuchi (1963) Tabuchi (1963) Yamanaka (1964)

10-2

2x to 2 x 10"

becomes smaller when the content of allophane decreases (Maeda and Warkentin, 1975). This indicates that dried samples have a larger proportion of large voids, i.e., that the small voids are lost preferentially on drying (Misono et al., 1953). The common observation that dried allophane soils resemble sands in their physical properties is due to formation of aggregates with small internal porosity and hence large interaggregate void volume. Tada (1965) described non-Darcy flow of water in fresh allophane soil. The conductivity was constant to hydraulic gradients of about 15, then increased about 20 times t o gradients of 50, after which the conductivity again decreased. Dried allophane soil did not show this effect. Iwata (1 963, 1966) published a thorough study of water movement in unsaturated soils t o clarify water redistribution and field capacity concepts. He found that allophane soils had a much higher unsaturated hydraulic conductivity than soil with crystalline clay minerals when compared at the same suction. For this reason the field capacity occurred at a higher suction in allophane soils. Iwata (1963) measured unsaturated hydraulic conductivity values of 0.1 cm day-' at 0.1 bar and 1 cm day-' at 0.05 bar for an allophane soil. These values were 3 to 4 times higher than measurements on an alluvial soil. Maeda and Warkentin (1975) measured values of 0.01 cm day-' at 1 bar and cm day at 7 bar. The unsaturated conductivity of allophane soils was higher than that of crystalline clay soils at suctions below 2 bar. The advance of the wet front during infiltration also increases from wet to dry allophane samples (Maeda and Warkentin, 1975). This is opposite t o the effect for crystalline clay minerals. The diffusivity was four orders of magnitude larger

252

T. MAEDA ET AL.

for the dry sample. The change in soil water diffusivity with change in bulk density was about the same as for soils with crystalline clay minerals. El-Swaify and Swindale (1968) found that relatively high levels of salinity and sodium in irrigation water could be used on allophane soils and still maintain adequate permeability. The sodium caused only slight changes in structure of the soil. El-Swaify (1973) found anion effects on hydraulic conductivity for allophane soils.

D. FIELD STUDIES ON INFILTRATION AND EVAPORATION

High percolation rates in the surface and subsoil present a problem in managing rice paddies in Japan. A percolation rate of 3-4 cm day-' is desired. Often bentonite is used to decrease percolation rates because soil compaction by rolling is not sufficient. Another difficulty is the variability of percolation rates within a field; percolation does not follow a normal distribution and a few high values can result in ratios of the mode to the mean of 0.05 to 0.3 (Ishikawa et d.,1963). This makes effective bentonite dressings difficult to achieve. Birrell (1952) comments that the natural variation in water content and compaction characteristics of allophane soils in the field has made engineering investigations difficult. The variability of physical properties of allophane soils in the field has received considerable attention. A number of Japanese research workers cooperated in a study organized by the Japan Society for Irrigation, Drainage, and Reclamation Engineering on field variability of physical properties (e.g., Kuroda, 1971, Tokunaga and Sato, 1975). Forsythe (1975) has reviewed measured infiltration rates for allophane soils in Central America. The values are generally high, initial infiltration rates of 20-70 cm hour-' and 2-hour rates of 5-20 cm hour-' are reported. He points out that these high rates make the soils unsuitable for furrow or flood irrigation. Nakano et al. (1970), in a series of three papers, reported laboratory measurements of infitration and evaporation from columns of layered volcanic ash soils of different grain size, and field measurements of water movement and evaporation. Internal soil drainage can often be increased by mixing the layers to overcome boundary effects (Kon, 1967).

E. WATER AVAILABLE FOR PLANT USE

Relatively little information has been published on field studies of water available to plants in allophane soils. Many numbers for available water are based on water held between 15-bar and 0.5- or 0.3-bar suction. There is evidence that

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

253

15 bar is too high for the upper limit; that 5- to 8-bar suction is observed in the field (e.g., Misono and Terasawa, 1957; Masujima and Mori, 1962). It is also to be expected that the lower limit may be closer to 0.1 bar for allophane soils with relatively high permeability. Kira et al. (1963) found the field capacity t o be at 0.08 to 0.1 bar, and commented on the difficulty of estimating field water regime from water-retention curves. Chichester er al. (1969) found the field capacity occurred at suctions of less than 0.05 bar for a pumice soil in Oregon. Youngberg and Dyrness (1964) found the value was below 0.1 bar. Shiina and Takenaka (1961) used the water content after 24 to 48 hours drainage as the upper limit. They found that crop growth decreased markedly when the suction exceeded 1.5 bars in the root zone. They also found considerable water movement up from wet subsoil layers, enough to supply 50% of evapotranspiration in their experiment. Masujima and Kon (1963) also found that water movement up from the subsoil contributed to available water. Shiina (1963) rejected the use of 0.5- and 15-bar suctions to define the available water. He said this must be based upon growth period and weather conditions as well as soil suction. Plants use water held at suctions as low as 0.03 bar, and initial wilting can occur at 2 bar. Masujima and Mori (1962) noted that water was not equally available over the range of available water, and that availability depended upon plant and soil factors. Masujima and Mori (1 962) studied physical properties and plant growth; increasing noncapillary porosity was associated with decreasing water availability. Optimum porosity for growth of beans was 30% at 0.5 bar and 20% at 0.03 bar. They suggested that land improvement could be achieved by mixing in different particle sizes to achieve an optimum void size distribution. Forsythe et al. (1964) contains a good summary review of measured physical properties such as bulk density and available water content. They rate available water in many allophane soils as average to low, although some allophane soils have a high available water content. On a volume basis the available water in allophane soils is not markedly different from soils with crystalline minerals (Swindale, 1964). On a weight basis, the numbers are very high because of the low bulk density of allophane soils. Bonfils and Moinereau (1971) report relatively high values of available water (15 to 21%), with the available water increasing with increase in organic matter content. V. Soil Engineering

The term “cohesive volcanic ash soils” is often used in the soil engineering literature. Cohesion indicates clay properties. The term allophane soils is used in this review in the same sense, and will, therefore, be used in this section as well.

254

T.MAEDA ET AL.

Many of the engineering properties of allophane soils in Japan have been studied on “Kanto loam,” an allophane soil found on the Kanto plain north of Tokyo. The subsoil, especially, of the Kanto loam shows typical allophane properties.

A. COMPACTION

Soils are compacted when large earth-moving equipment is employed in land reclamation. From the viewpoint of engineering, it is important to know the compaction characteristics of soils. The compaction curve for a soil is a plot of water content versus the bulk density whch can be achieved by a specified compactive effort at that water content. This curve shows a maximum bulk density at a water content called the “optimum water content” for Compaction. Compaction produces a moderate increase in density and a large increase in strength of allophane soils. However, on resaturation the strength is again decreased (Northey, 1966). AUophane soils have a remarkably high natural water content compared with soils containing crystalline clay minerals, and moreover water-holding characteristics change during drying. These differences are reflected in differences in compaction characteristics. Allophane soils have low maximum bulk densities, in the range 0.8-1.3 g cmd3and relatively high optimum water contents (Table v>. The optimum water content is much below the natural water content. The compaction curve of a soil with crystalline clay minerals is the same regardless of initial water content at the beginning of the compaction test; however, allophane soils show different curves depending upon the initial water content and the amount of remolding produced during testing (Birrell, 1951). An undried allophane soil does not show a distinct maximum in bulk density, TABLE V Compaction Characteristics of Two Typical Allophane Soils

Sample Kanto loam -Natural -AU-dry -Oven-dry Java andosol -Natural -&-dry -Oven-dry

Maximum dry density 1 (g cmW3

Optimum water content (w, %)

0.69 0.79 0.86

105 86

0.55 0.69 0.79

127 95 80

74

Reference

Kuno and Mogami (1949) Kuno and Mogami (1949) Kuno and Mogami (1949) Wesley (1973) Wesley (1 973) Wesley (1973)

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

255

and hence no identifiable optimum water content. Since the natural water content exceeds the optimum water content, this curve can be measured only by gradually drying samples for compaction. The bulk density increases only gradually as the water content is decreased. Once’the soils are dried, they show typical compaction curves. This behavior is illustrated in Fig. 4, which is typical of the results obtained. These characteristics are described by Kuno and Mogami (1949), Birrell (1951)’ Tada (1965), Tokunaga (1965), Northey (1966), Frost (1967)’ Takenaka (1973), Wesley (1973), Adachi and Takenaka (1973)’ and others . The compaction curve obtained by first drying and then rewetting a sample, therefore, forms a loop with only the drying portion showing a maximum density (Takenaka, 1973). This is due t o the reduction in the amount of water retained at any suction value. This loop forms when the soil is dried below a critical water content, which Tada (1965) found to be at a suction of 15 bar for the Kanto loam subsoil. This is the highest suction which the subsoil would be subjected to under natural soil conditions with drying only by plant roots. Since it is often difficult to find the optimum water content and the maximum dry density on the compaction curve of fresh allophane soil, the criterion of maximum dry density cannot be applied for embankment work. The degree of saturation is used in Japan in place of the maximum dry density to follow the degree of compaction of allophane soil (Kuno and Yabe, 1960, 1962). The permeability of compacted soils is an important engineering consideration. When allophane soils are compacted, with a decrease in water content, the dry density increases slightly, but the permeability also increases. According to Tada’s (1965) experiments, coefficient of permeability reaches a minimum near the natural water content, and tends to gradually increase on lowering of water content. Tokunaga’s (1969) experiments studied these phenomena in relation to

Zero air voids

-----.

<#--

-\

40 0

35 50

60

70 80 Water Content

90

100

Yo

FIG. 4. Compaction curve for allophane soil on gradual drying (from Northey, 1966). A, Dried from natural water content (both samples, 7592B and 7592C); B, dried to 53% and rewet (7592B); C, dried to 70% and rewet (7592C).

256

T. MAEDA ET AL. TABLE VI Permeability of Compacted Allophane Soils‘

Water content (%)

Permeability (cm sec-’ )

150 100

7 x lod 10-5

50

lo-*

Bulk density (g ~ m - ~ ) 0.5

0.65 0.75

*

‘From Tokunaga (1969).

consistency and structure of the soil. Microscopic observations revealed that in the range of low water content the soil has a granular structure with large voids. Therefore, it has a high permeability coefficient in spite of the higher bulk density. As the water content increases near the plastic limit, aggregated blocks are developed causing hindrance to water passage and lowering permeability. Further increase of water content up to around the natural water content causes the aggregated blocks to flow into a pastelike structure. In this range of water contents the coefficient of permeability is a minimum, and the dry density is low because of high water content (Table VI). Allophane soils rich in humus show a different behavior. The organic matter forms stable humus which combines with soil particles forming an aggregated structure. Permeability increases slightly at high water content for compacted soil.

B. STRENGTH

Allophane soils can be stable in the undisturbed state, often occurring in relatively steep banks (Northey, 1966; Wesley, 1973). They are also relatively resistant to erosion, although landslides and water erosion occur on steep slopes. The strength of allophane soils disturbed by excavation and embankment is remarkably lower than that of the undisturbed soils. Once disturbed, allophane soils are too weak t o ensure traffcability for construction equipment (Highway Research Board, 1973). The Kanto loam has the bearing capacity to support buildings of four or five stories in spite of its high natural water content. The unconfined compressive strength of Japanese allophane soils is in the range of 1.O-2.3 kg cm-’ (Highway Research Board, 1973). This is more than five times the strength of alluvial nonvolcanic soils at the same water content. It is also recognized that the bearing capacity, estimated from the N-value obtained from the standard penetration test, is higher than that of the nonvolcanic clays. For allophane soils in the

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

257

undisturbed condition, the experimental equation between the N-value of the standard penetration test and the bearing capacity (4a) is given as follows: 4a = (2 to 2.5)N For alluvial clay soils the equation is: qa = (1 to 1.3)N

Birrell (1951) found small f,riction angles, 0-8", and low shear strength, 0.2-0.4 kg cm-', from triaxial test results. Pope and Anderson (1960) measured values as low as 1" for friction angle and 0.5 kg cm-' for the cohesion parameter. Wesley (1973) found undrained shear strength values of 1.0 to J.2 kg cm-2, and in situ vane shear results of 0.7 to 1.0 kg cm-2. The unconfined compressive strength of allophane soils decreases with increasing organic matter content (Yamanouchi and Yasuhara, 1972). Most authors comment on the large variability over short distances in engineering properties of allophane soils (Pope and Anderson, 1960; Northey, 1966; Wesley, 1973). This high heterogeneity in the field makes it difficult t o use measured results in design of earth structures. Another feature of allophane soils is that strength does not increase with depth, i.e., with increasing overburden pressure. Since it is not possible to estimate strength from the natural water content, no practical indices exist to estimate the strength. The strain at failure is only 2-3% for allophane soil, against 2-6% for other clay soils. When allophane soils are disturbed, the compressive strain increases (Gradwell and Birrell, 1954; Takenaka, 1965). Gradwell and Birrell (1954) and Komamura and Takenaka (1973) show the Mohr circles for shearing resistance as a function of stress for undisturbed and remolded allophane soils. Allophane soils have moderate measured sensitivity, the ratio of undisturbed to remolded shear strength. Birrell (1951) gives values of 6-12, Wesley (1973) measured values between 1 and 3 . Wells and Furkert (1972) showed that water in undisturbed allophanes was held in hydrogen-bonded clusters. Remolding breaks up these clusters, and the water becomes distributed as single-linked molecules. Mophane soils have high water contents and a well-developed soil structure. The lowering of the strength on remolding is due to change in water-holding characteristics and structure peculiar t o allophane soils. On disturbance, water which was held firmly in the voids is released and free to flow. The measured soil suction has decreased; the decrease is largest at low suction values. In consequence, the soils soften and lose strength. The soils least resistant t o this softening are buried soils with high organic matter, especially from old volcanic ash layers (Takenaka, 1966; Komamura and Takenaka, 1973; Takenaka and

258

T. MAEDA ET AL.

Yasutomi, 1965). The changes in stress and pore pressure with time of remolding or kneading are shown in Table VII. The data are taken from Takenaka and Yasutomi (1965). They review the changes in soils on remolding, which can lead to either softening or hardening: (1) the structure units are forced together, leading to decrease in suction; (2) the units are separated, leading to increased suction; (3) water in voids is released, giving decreased suction; (4) the structure unit is broken, exposing new surfaces which absorb water, leading to increase in suction; (5) absorbed water is released, leading to decreased suction. Depending upon the net result of these changes, either hardening or softening can occur. The usual case for allophane soils is softening on remolding. Adachi and Takenaka (1973) measured the increase in unconfined compressive strength with increasing soil suction. The relationship was linear on a log-log plot for samples in which the suction was increased by gradually decreasing the water content during remolding. However, when samples were dried t o different water contents before remolding, strength did not increase beyond a suction of about 1 bar. If disturbed soils whose strength has been decreased by remolding are kept at the same water content, the strength is gradually recovered with time. This strength regain is one of the features of allophane soils; it is often called “thixotropy” (Takenaka and Yasutomi, 1965; Komamura and Takenaka, 1973; Yasutomi, 1974). Table VIII shows typical results for strength regain. The soils which show little strength regain are thought to soften by structure units being forced together and releasing water. No subsequent movement apart of units takes place. The large strength regain is shown by soils in which water is released from within the structure on remolding. This water can be reabsorbed by the units on standing, and hence the soil hardens. Wells and Furkert (1972) describe the sensitivity test used t o recognize

TABLE VII Strength and Pore Pressure Changes due to Remolding for Two Allophane Soil% Remolding time (minutes)

0 2 10 20 40

Stress (kg cm-’)

Suction (millibar)

A

B

A

1.1

0.6

150

90

-

0.8

10 10

110

0.75 0.6

-

0.6 0.2

0.15

‘From Takenaka and Yasutomi (1965).

B

50 -

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

259

TABLE VIII Strength Regain After Remolding for Two Allophane Soils' Time (days)

1 10 100

Cohesion (kg cm-2)

Friction angle (degrees)

A

B

A

B

0.15 0.19 0.20

0.29 0.30 0.41

1.8 1.8 2.0

12.0 12.8 13.3

aFrom Komamura and Takenaka (1973).

allophane soils in the field in New Zealand. When pressed between thumb and fingers under increasing pressure the soil suddenly shears, releasing free water. Fujioka et al. (1965) report that polyvinyl alcohol soil stabilizers increase the strength of allophane soils, decrease swelling, and increase the maximum dry density. Umeda and Nagasawa (1974) found a decreased shear strength and decreased soil suction after freezing an allophane soil.

C. CONSOLIDATION

The e-log p curves of allophane soils have been obtained by a number of workers ( e g , Gradwell and Birrell, 1954; Suzuki, 1973). Allophane soils are fairly compressible, once the preconsolidation pressure has been exceeded. Fieldes and Claridge (1975) quote values, from New Zealand studies, of 2.4 to 3.1 mz yr-' for coefficient of consolidation. This high value is due to the aggregation of the clay-size grains. The initial void ratio of allophane soils is much larger than for soils with crystalline clay minerals, from 2 to 3.5 for low humic allophane and 6 to 7 for allophane soils with high humus content. The e-log p curve of undisturbed allophane soils shows a clear flex point, so it is easy to find the preconsolidation pressure. For the Kanto loam in Japan and for New Zealand soils, the values range from 1 t o 3 kg cm-', but are not correlated with depth. The curve for disturbed samples shows a smooth decrease in void ratio, and it is difficult to obtain a preconsolidation load (Highway Research Board, 1973). The preconsolidation pressure often exceeds the overburden pressure due t o the strength of the aggregates (Gradwell and Birrell, 1954). Allophane soils show a large secondary consolidation, which decreases as the applied load increases (Birrell, 1951; Northey, 1966). This is due to breakdown

260

T. MAEDA ET AL.

of the structure units. Secondary consolidation increases with increasing organic matter content of the soil (Yamanouchi, 1965). The observed rebound for undisturbed and for remolded allophane soils on removing the load is remarkably small. Thus the elastic compression is very small compared with the total consolidation. Initial settlement, secondary consolidation, and coefficient of compressibility are decreased by remolding allophane soils.

D. SOIL STABILIZATION

Soil stabilization is necessary in order to use allophane soils, especially with high organic content, for highways or earth dams. Results on stabilization of highly organic allophane soil obtained by Yamanouchi (1963) were as follows: Portland cement is not useful for stabilization because calcium is absorbed by the organic matter, and hardening of cement is hindered. Addition of calcium chloride, quick hardening cement, or calcium oxide is useful for stabilization. Arizumi (1962) found that hydrated gehlenite (2Ca0, N 2 O 3 ,SiOz .nH2 0) was formed by mixing Kanto loam with calcium hydroxide or calcium sulfate. The effect of asphalt emulsion is not dependable nor is a high density obtained, because of the high amount of water added to facilitate mixing. The combined use of asphalt emulsion and Portland cement gave better results than asphalt emulsion alone (Yamanouchi, 1963). Dispersing agents, such as calcium lignosulfonate, and aggregating agents, such as copolymerized vinylacetate and maleic acid, increased soil strength. Soil stabilization of organic allophane soil is best achieved with lignin materials such as spent sulfite liquor or its extract, with potassium dichromate, aluminum sulfate, or ferric chloride as the auxiliary agent. Northey and Schafer (1974) developed methods of testing the effect of adding lime to wet allophane soils, and found increased soil strength additions of about 5%lime.

E. ADHESION AND COHESION

Adhesion of allophane soil to a metal surface is about one-quarter of that measured for a soil with crystalline clay minerals (Yamanaka, 1964). Adhesion is low at low water content, then quickly increases to a maximum as water content is increased. Cohesion of allophane soil is low at zero water content, then increases to a maximum before decreasing again. Soils with crystalline clay minerals have a high cohesion at zero water content, which decreases rapidly as water content

PHYSICAL PROPERTIES OF ALLOPHANE SOILS

261

increases (Yarnanaka, 1964). The cohesion of an air-dried allophane soil is lower than that of an undried soil (Yamanaka, 1964; Maeda and Soma, 1974).

REFERENCES

Adachi, T., and Takenaka, H. 1973. Trans. Jpn. SOC. Irrig. Drain. Reclam. Eng. (Nogyo Doboku Gakkai Ronbunshu) 43,26-32. Ahmad, N., and Prashad, S. 1970. J. Soil Sci. 21,63-71. Aomine, S., and Egashira, K. 1970. Soil Sci. Plant. Nutr. (Tokyo) 16, 204-211. Arizumi, A. 1962. Rep. Jpn. Exp. Stn., Civ. Eng. 110. Baba, H. 1971. J. Fac, Agric.’Iwate Univ. (Iwate Daigaku Nogakubu Hokoku Univ.) 10, 283-291. Binell, K . S. 1951.Proc. Congr. R. SOC.N.Z., 7th pp. 208-216. Birrell, K. S. 1952. Proc. Aust. N.Z. Conj: SoilMech. Found. Eng., 1st pp. 30-34. Birrell, K. S. 1966. N.Z. J. Agric. Res. 9, 554-564. Birrell, K. S., and Fieldes, M. 1952. J. Soil Sci. 3, 156-166. Bonfils, P., and Moinereau, J. 1971. Cah. ORSTOM, Ser. Pedol. 9, 345-363. Borchardt, C. A., Theisen, A. A., and Harward, M. E. 1968, Soil Sci. SOC.Am., Proc. 32, 735-737. Brewer, R. 1964. “Fabric and Mineral Analysis of Soils.” Wiley, New York. Chichester, F. W.,Youngberg, C. T., and Harward, M. E. 1969. Soil Sci. Sue. Am., Proc. 33, 1 15-120. Cochran, P. H., Boersma, L., and Youngberg, C. T. 1967. Soil Sci Soc. Am., Proc. 31, 454459. Colmet-Daage, F., and Cucalon, F. 1965. Fruits 20, 19-23. Colmet-Daage, F., Cucalon, F., Delaune, M., Gautheyrou, J., Gautheyrou, M., and Moreau, B. 1967. Cah. ORSTOM, Ser. Pedol. 5,l-38. Colmet-Daage, F., Gautheyrou, J., Gautheyrou, M., de Kimpe, C., Sieffermann, G., Delaune, M., and Fusil, G. 1970. Cah. ORSTOM, Ser, Pedol. 8,113-172. Colmet-Daage, F., Gautheyrou, J., Gautheyrou, M., de Kimpe, C., and Fusil, G . 1972. Cah. ORSTOM, Ser. Pedol. 10, 169-191. Croney, D., and Coleman, J. D. 1954. J. Soil Sci. 5.75-84. Davies, E. B. 1933. N.Z. J. Sci. Technol. 14, 228-232. Egashira, K., and Aomine, S. 1974. Clay Sci. 4, 231-242. El-Swaify, S. A. 1973. Soil Sci. 115,64-72. El-Swaify, S. A., and Swindale, L. D. 1968. Trans. Int. Congr. Soil Sci., 9th 1, 381-389. Espinoza, W., Rust, R. H., and Adams, R. S., Jr. 1975. Soil Sci. SOC. Am., Proc. 39, 556-561. Fieldes, M., and Claridge, G. G. C. 1975. I n “Inorganic Components” (J. E. Gieseking, ed.), Soil Components, Vol. 2, pp. 351-393. Springer-Verlag, Berlin and New York. Flach, K. W. 1964. Proc. Panel Volcanic Ash Soils Latin Am., Turrialba, Costa Rica Pap. A.7. Forsythe, W. M. 1972. Panel Volcanic Ash Soils Am., Pasto, Colombia pp. 481-495. Forsythe, W. M. 1975. Proc. Soil Manage. Trop. Am., North Carolina State Univ. pp. 155-167. Forsythe, W. M., Gavande, S. A., and GonzAlez, M. A. 1964. Proc. Panel Volcanic Ash Soils Latin Am,, Turrialba, Costa Rica Pap. B.3. Frost, R. J. 1967. Proc. Southeast Asian Reg. Conf: Soil Eng., Ist, Bangkok pp. 44-53.

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Fujioka, Y., Nagahori, K., and Sato, K. 1965. Trans Jpn. Soc. Irrig. Drain Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu) 10,1-6. Fujiwara, H., and Baba, N. 1973. Trans. Jpn. Soc. Irrig. Drain. Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu 48,29-33. Galindo-Griffith, G. G. 1974. Ph.D. Thesis, Univ. of California, Riverside. Gautheyrou, J., Gautheyrou, M., and Colmet-Daage, F. 1976. “Chronobibliographie des Sols h Allophane.” ORSTOM, Cent. Antilles. Gradwell, M., and Birrell, K. S . 1954. N Z . J. Sci. Technol., Sect. B 36, 108-122. Grim, R. E. 1953. “Clay Mineralogy.” McGraw-Hill, New York. Higashi, A. 1951. J. Fac. Sci., Hokkaido Univ., Ser. 2 4, 21-29. Higashi, A. 1952. J. Fac. Sci., Hokkaido Univ., Ser. 2, 4, 95-107. Highway Research Board. 1973. “On the Earthworks of Kanto Loam,” pp. 78-1 14. Kyorits Shyuppan Press, Tokyo. Hughes, I. R., and Foster, P. K. 1970. N Z . J. Sci. 13, 89-107. Ikegami, M., and Tachiiri, M. 1966. Rec. Land Reclam. Res. 1 5 , 3 7 4 1 . Ishikawa, T., Tokunaga, K., and Tsukidate, K. 1963. J. Agric. Eng. Soc. Jpn. 31, 22-30. Ito, M. 1964. Trans, Jpn, SOC. Irrig. Drain. Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu) 9, 1 4 . Iwata, S. 1963. J. Agric. Eng. Soc. Jpn. 30, 3845-394. Iwata, S . 1966. Bull. Natl. Inst. Agric. Sci., Ser. 13 16, 149-176. Kanno, I, 1961. Bull. Kyushu Agric. Expt. Sta. (Kyushu Nogyo Shikenjo Iho) 7, 61-73. Kasubuchi, T., 1975a. Soil Sci. Plant Nutr. (Tokyo) 21, 73-17. Kasubuchi, T. 1975b. SoilSci. Plant Nutr. (Tokyo) 21, 107-112. Kawaguchi, K., Kita, D., and Mori, H. 1963. J. Sci. Soil Manure, Jpn. 34, 7-12. Kira, Y., Ambo, F., SBma, K., and Ito, K. 1963. Trans. Jpn. SOC.Irrig. Drain. Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu) I, 76-80. Kitagawa, Y. 1971. A m . Mineral. 56,465-475. Kobo, K. 1964. F A 0 World Soil Res. Rep, 14, pp. 71-73. Kobo, K., and Oba, Y. 1964. In “Volcanic Ash Soils in Japan,” pp. 30-31. Min. Agric. For., Japan. Kodani, Y., Kono, H., and Uchida, K. 1976. Trans. Jpn. SOC. Irrig. Drain. Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu 60, 7-13. Komamura, M., and Takenaka, H. 1973. J. Agric. Sci. Tokyo Univ. 11, 331-341. Kon, T. 1967. Res. Corresp. Soil Plant Growth Hokkaido 58, 28-50. Kubota, T. 1971. J. Clay Sci. Soc. Jpn. 11, 73-84. Kubota, T. 1972. Soil Sci. Plant Nutr. (Tokyo) 18, 79-87. Kuno, G., and Mogami, T. 1949. Technol. Eng. Rep., Tokyo Univ. 3, 7, 8. Kuno, G., and Yabe, M. 1960. Tech. Rep. Civ. Eng. J p n 4, 3 3 4 1 . Kuno, G., and Yabe, M. 1962. Tech. Rep. Civ. Eng. Jpn. 6, 15-24. Kuroda, M. 1971. Trans. Jpn. Soc. Irrig. Drain. Reclam. Eng. (Nogyo Doboku Gakkai Ronbunshu) 36,14-20. Maeda, T. 1968. J. Hokkaido Branch Agric. Meterol. Jpn. 19,61-72. Maeda, T., and Soma, K. 1974. Soil Phys. Cond. Plant Growth, J p n 30, 15-22. Maeda, T., and Warkentin, B. P. 1975. SoilSci. SOC.Am., Proc. 39, 398-403. Maeda, T., Sasaki, S., and Sasaki, T. 1970. Trans. Jpn. Soc. Irrig. Drain. Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu) 31,25-28. Maeda, T., Soma, K., and Sasaki, S. 1976. Trans, Jpn. SOC.Irrig. Drain Reclam. Eng. 61,9-11. Masujima, H . 1962. Res. Bull. Hokkaido Natl. Agric. Exp. Stn. 17,40-41. Masujima, H., and Kon, T. 1963. Res. Bull Hokkaido Natl. Agric. Exp. Stn. 80, 70-76. Masujima, H., and Mori, T. 1962. Res. Bull. Hokkaido Natl. Agric. Exp. Stn. 79, 1-35.

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Misono, S., and Terasawa, S. 1951. Bull. Natl. Inst. Agric. Sci,, Ser. B 7, 11-103. Misono, S., Terasawa, S., Kishita, A., and Sudo, S. 1953. Bull. Natl. Inst. Agric. Sci., Ser. B 2,95-124. Miyazawa, K., and Konno, T. 1916. Res. Bull. Hokkaido Natl. Agr. Exp. Stn. 114, 89-118. Nagata, N. 1963. Trans. Agric, Eng. SOC.Jpn. 7, 31-42. Nakano, M., Tabuchi, T., and Yawata, T. 1970. Trans. Jpn. SOC.Irrig. Drain Reclam. Eng. (Nogyo Doboku Gakkai Ronhunshu) 31, 11-24. Northey, R. D. 1966. N.Z. J. Sci. 9, 809-832. Northey, R. D., and Schafer, G. J. 1914. N.Z. J. Sci. 17, 131-150. Oba, Y., and Kobo, K. 1965. J. Sci. Soil Manure, Jpn. 36, 203-206. Packard, R. Q. 1951. Soil Sci. 83, 213-289. Parfitt, R. L., and Scotter, D. R. 1912, Papua N e w Guinea Agric. J. 23, 9-11. Pope, R. J., and Anderson, M. W. 1960. Am. SOC. Civ. Eng. Res. Con5 Shear Strength Cohesive Soils, Univ. Colorado, Boulder pp. 3 15-340. Rousseaux, J. M., and Warkentin, B. P. 1916. Soil Sci. SOC.Am., J. 49,446-451. Sasaki, S. 1951. J. Sci. SoilManure, Jpn. 28, 11-21. Sasaki, T., Maeda, T., and Sasaki, S. 1969. Trans. Jpn. SOC. frrig. Drain. Reclam. Eng. (Nogyo Doboku Gakkai Ronbunshu) 27,Sl-60. Schalscha, E. B., Gonzalez, C., Vergara, I., Galindo, G., and Schatz, A. 1965. Soil Sci SOC. Am, Proc. 29,481-482. Sherman, G. D. 1951.Science 125,1243. Sherman, G. D., Matsusaka, Y., Ikawa, H., and Uehara, G. 1964. Agrochimica 8, 146-163. Shiina, K. 1963. Bull. Agric. Eng. Res. Stn. 1, 83-156. Shiina, K., and Takenaka, H. 1961. Trans Jpn. SOC. Irrig. Drain. Reclam Eng (Nogyo Doboku Gakkai Ronhunshu) 2,49-55. Soma, K., and Maeda, T. 1914. Trans. Jpn. SOC.Irrig. Drain. Reclam Eng. fNogyo Doboku Gakkai Ronhunshu) 49,21-34. Sowers, G. F. 1965. In “Methods of Soil Analysis, Part I” (C. A. Black, D. D. Evans, J. L. White, L. E. Ensminger, and F. E. Clark, eds.), Monogr. No. 9, pp. 391-399. Am. SOC. Agron., Madison, Wisconsin. Sudo, S . , and Suzuki, T. 1963. Trans. Agric. Eng. SOC.Jpn. 7, 104-108. Suzuki, A. 1913. Bull. Fac. Eng. Kumamoto Univ. 100, 1-33. Swindale, L. D. 1964. Proc. Panel Volcanic Ash Soils Latin Am., Turrialha, Costa Rica Pap. B.lO. Tabuchi, T. 1963. Trans. Agric. Eng. SOC.Jpn. 7,32-31. Tabuchi, T., Tabuchi, K., and Nagata, N. 1963. Trans. Agric. Eng. SOC.Jpn. 7, 53-60. Tada, A. 1965. Trans Jpn. SOC.Irrig. Drain. Reclam Eng. (Nogyo Doboku Gakkai Ronhunshu) 1 4 , 4 1 4 5 . Tada, A., and Yamazaki, F. 1963. Trans. Jpn. SOC. Irrig. Drain. Reclam. Eng. (Nogyo Dohoku Gakkai Ronhunshu) 5,11-23. Takenaka, H. 1961. Rec. Land Reclam. Rex 12, 23-21. Fac. Agric., Univ. Tokyo. Takenaka, H. 1965. Trans. Agric. Eng. SOC,Jpn. 14,32-35. Takenaka, H. 1966. Soil Phys. Cond. Plant Growth, Jpn. 14, 21-25. Takenaka, H. 1973. Trans. Jpn. SoilMech. Found. Eng. No. I80 21, 13-19. Takenaka, H., and Yasutomi, R. 1965. Trans. Agric. Eng. SOC.Jpn. 14,54-59. Takenaka, H., Tabuchi, T., Tabuchi, K., and Tada, A. 1963. Trans. Agric. Eng. SOC.Jpn. 7, 61-61. Terasawa, S. 1967. Bull. Natl. Inst. Agric. Sci., Ser. B 19, 197-228. Tokunaga, K. 1965. In “Soil Physics” (F. Yamazaki, ed.), pp. 248-260. Yokendo Press, Tokyo.

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Tokunaga, K. 1969. ‘Soil Physics.” Yokendo Press, Tokyo. Tokunaga, K., and Sato, T. 1975. Trans Jpn. SOC. Irrig. Drain. Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu) 55, 1-8. Tsujinaka, N., Sasaki, T., Maeda, T., and Sasaki, S. 1970. J. Fac. Agric., Hokkaido Univ. 56, 267-291. Umeda, Y., and Nagasawa, T. 1974. Trans. Jpn. SOC. Irrig. Drain Reclam Eng. (Nogyo Doboku Gakkai Ronbunshu) 54,6-10. van Schuylenborgh, J. 1953. Neth. J. Agric. Sci. 1,50-57. Wada, K., and Harward, M. E. 1974. Adv. Agron 26,211-260. Wada, K., and Henmi, T. 1972. Clay Sci. 4,127-136. Wada, K., Yoshinaga, N., Yotsumoto, H., Ibe, K., and Aida, S. 1970. Clay Miner. 8, 487489. Wada, S., and Wada, K. 1975. Annu, Meet. Clay Sci. SOC.Jpn. Warkentin, B. P. 1972. Can. J. Soil Sci. 52,457-464. Warkentin, B. P., and Maeda, T. 1974. Soil Sci SOC.Am., Proc. 38,372-377. Wells, N., and Furkert, R. J. 1972.Soil Sci. 113, 110-115. Wesley, L. D. 1973. Geotechnique 23,471494. Yakuwa, R. 1943. M e m Sapporo Meterol. Obsew. 2(2), 41-104. Yamanaka, K. 1964. In “Volcanic Ash Soils in Japan,” pp. 69-75. Min. Agric., Japan. Yamanouchi, T. 1963. Proc. Asian Reg. ConJ Soil Mech. Found. Eng., 2nd, T o k y o 1, 35 9-363. Yamanouchi, T. 1965. Annu. Rep., Jpn. Road Assoc. pp. 1-21. Yamanouchi, T., and Yasuhara, K. 1972. Trans. Jpn. SOC.Civ. Eng. 13, 23-29. Yamazaki, F., and Takenaka, H. 1965. Trans. Agric. Eng. SOC.Jpn. 14,4648. Yasutomi, R. 1974. J. SOC,Rheol. Jpn. 2,53-57. Yazawa, M. 1976. Trans. Jpn. SOC. Irrig, Drain. Reclam. Eng. (Nogyo Doboku Gakkai Ronbunshu 658-14. Youngberg, C. T., and Dymess, C. T. 1964. Soil Sci 97,391-399.

DISEASE AND INSECT RESISTANCE IN RICE' Gurdev S. Khush International Rice Research Institute, Los Bafios, Philippines

..................................................

1. Introduction 11. Disease Resistance

111.

. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . A. Fungal Diseases . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . , . . . . . . . . B. Bacterial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virus Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insect Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Stem Borers ................................................ B. Plant Hoppers ...............................................

C. Leafhoppers ................................................ D. GallMidge ................................................... IV. Developing Varieties with Multiple Resistance . . . . . . . . . . . . . . . . . . . . . A. Breeding Methods and Procedures . . . . . . . . . . . . . . . . . , . . . B. International Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . V. Stability of Resistance . . . . . . . . . . . . . . . . . .. . . . ... .. .. A. Vertical Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Horizontal Resistance . . . . . . . . . . . . . . . . . . . . .. . . VI. Conclusions ................................................... References ....................................................

. . .. .. .. . .. . . .. . .. . . . . . . .. . . .. .. ... ... . . . . .. .. .. . . .. .. . . . . . .. . . . ..

265 266 266 219 287 301 301 306 3 16 318 322 3 24 3 28 3 29 330 332 333 333

I. Introduction

During the last 10 years major changes have occurred in the varietal composition of and cultural practices for rice. High-yielding varieties are now planted on approximately 25% of the 130,000,000hectares (ha) planted to rice all over the world. These varieties are characterized by early maturity, photoperiod insensitivity, short stature, high tillering, and dark-green erect leaves. About 100 improved plant-type varieties have replaced hundreds of tall traditional cultivars. The genetic variability of the crop is thus reduced. After the introduction of varieties with improved plant type, farmers started using improved cultural practices, such as better water and weed control, higher rates of fertilizers, and higher plant populations per unit area. The development of irrigation facilities and the availability of early maturing, photoperiod-insensitive varieties have enabled the farmers in tropical Asia to grow successive rice crops throughout the year in large areas. 'This paper was prepared when the author was Visiting Professor of Agronomy at Colorado State University, Fort Collins, Colorado.

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Reduced genetic variability, improved cultural practices, and continuous cropping with rice-factors for increased rice production-have increased the genetic vulnerability of the crop. Within the last few years serious outbreaks of diseases and insects harmful to rice have occurred in several countries. Very little research has been done on the chemical control of rice diseases in the tropics. Several insecticides have been identified, but chemical control of high insect populations for prolonged periods under tropical climate-where insect generations overlap throughout the year-is very expensive. Social and economic conditions in the tropics present other obstacles to the chemical control of rice diseases and insects. Research on the control of diseases and insects through host resistance has been increasingly emphasized in recent years. All international as well as national rice improvement programs devote major shares of their efforts to incorporating into their breeding materials genetic resistance t o major diseases and insects. A large number of pathologists, entomologists, and breeders are engaged in t h s endeavor throughout the rice-growing areas of the world. As a result, improved varieties with multiple resistance to several diseases and insects are now available to rice growers. Those varieties and the others still in the pipeline will play crucial roles in increasing the world’s food production. This chapter reviews the progress made in developing rice that is resistant to diseases and insects. The topics t o be discussed include the nature of the disease or insect, its distribution, genetic variability of the pathogen, host resistance, genetics of resistance, and breeding for resistance. The review emphasizes recent work; it includes references to previous reviews on each subject. II.

Disease Resistance

Numerous diseases of rice, caused by fungi, bacteria, viruses, and nematodes, have been recorded in different rice-growing areas of the world. Some diseases occur wherever rice is grown: some are of both regional and international importance, others occur in local areas. Some diseases reach epidemic proportions and cause serious crop losses, while others cause only negligible crop losses. This chapter deals with only diseases of international importance which cause considerable crop losses. For information on diseases not covered here, the reader is referred to the excellent treatment of the subject by Ou (1972).

A. FUNGAL DISEASES

Most rice diseases are caused by fungi. Among the 60 rice diseases discussed by Ou (1972), 37 are fungal diseases. Fungal diseases attack the plant foliage, stems,

DISEASE AND INSECT RESISTANCE IN RICE

267

roots, leaf sheath, or inflorescence, and grains. Some affect only one plant organ. Four fungal diseases of major economic importance are reviewed here.

I . Blast The blast disease of rice occurs in all rice-growing areas of the world. It is the most important disease of the rice plant and causes serious and sometimes total yield losses. It may infect the leaves, nodes, panicles, and other aerial parts of the plant. It is also known as leaf blast, node blast, panicle blast, or neck rot, depending upon the plant part affected. a. Variation in Pathogenicity. Rice blast is caused by Pyricularia oryzae, a highly variable organism, The differences in pathogenicity of the fungus strains were first recorded by Sasaki (1922, 1923). He noticed that rice varieties resistant to one strain were severely infected by another. Intensive investigations on pathogenic variability of the blast fungus in Japan were started in 1950 when some resistant rice varieties, such as Futaba, suddenly became susceptible. Around 1960, 12 varieties were selected as differentials and 13 pathogenic races were identified (Goto, 1965). Latterell et al. (1954) reported on pathogenic races of blast in the United States. With the use of additional isolates from Asia and Latin America, 15 races were identified (Latterell et aL, 1960). Pathogenic variability was also reported in India (Padmanabhan, 1965b), Taiwan (Chiu etal., 1965), Korea (Ahn and Chung, 1962; Lee and Matsumoto, 1966), and Colombia (Galvez and Lozano, 1968). More than 100 races have been identified in the Philippines (Bandong and Ou, 1966; Ou and Jennings, 1969), and the number continues to increase. Each country has used various sets of differential varieties in identifying blast races. Therefore, races identified in one country cannot be compared with races identified in other countries. A cooperative study was started in 1963 between Japan and the United States to develop an international set of differential varieties. Hundreds of isolates collected in Japan and the United States were tested during a 3-year study. Of 39 differential varieties, 8 were selected t o form an international set of differentials for blast. With the set, 32 race groups were characterized. The races were called international races and given the designation lA, lB, etc., to lH, followed by numbers (Atkins et aL, 1967; Goto et al., 1967). In the race studies, a pure culture is obtained by isolating a single conidium from a sample. Inoculum for testing pathogenicity is prepared from the pure culture. Ou and Ayad (1968) tested 56 monoconidial cultures from the same crop of spores of a leaf lesion on the Philippine set of differentials and found 14 races; 44 monoconidial cultures from a second lesion were differentiated into 8 races. They also found that 25 monoconidial subcultures from each of two single conidial pure cultures were differentiated into 9 and 10 races, respectively.

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Testing 4 varieties, Giatong and Frederiksen (1969) found 20 monoconidial lines that separated into 4 to 7 races. In three consecutive generations, the monoconidial lines continued to change into different races. Similar results were reported by Chien (1968) and Ou et al. (1971~). b. Varietal Resistance. Various methods for evaluating resistance to blast have been developed in different countries. To assess the disease reaction more accurately and to handle a large number of varieties in a short time, a uniform testing method was adopted for the International Blast Nursery Program (Ou, 1965a). The blast nursery method of testing allows quick evaluation of the resistance of a large number of rice varieties to a number of races in the region. According to Ito (1965), varietal differences in resistance to blast were observed as early as 1900; varieties Kameji and Aikoku were considered highly resistant, and Shinriki was thought susceptible. Numerous resistant varietiesmany of foreign origin-have since been identified in Japan and utilized in the breeding program (Toriyama, 1972). Blast-resistant varieties have been identified in India (Padmanabhan, 1965a), Thailand (Dasananda, 1965), Taiwan (Chang et al., 1965), and the United States (Atkins et al., 1965). Varietal reaction may vary from country to country, from locality to locality, and from season to season in the same locality. To identify blast-resistant varieties with a broad spectrum of resistance, the Working Party Meeting of the International Rice Commission (IRC) held in Sri Lanka in 1959 initiated the Uniform Blast Nurseries. During the 1963 symposium on the rice blast disease at the International Rice Research Institute (IRRI), the International Uniform Blast Nurseries (IUBN), initiated by FAO-IRC, were modified and strengthened, and leadership for coordination was assigned to IRRI (Ou, 1965a). A total of 258 selected varieties of rice identified on the basis of resistant reactions in initial tests were included in the IUBN. The results of nursery tests at 50 locations in 26 countries were reported by Dr. Ou and his colleagues in various issues of the International Rice Commission Newsletter. In 1966, an additional 321 varieties selected from the IRRI blast nursery were included in the international tests. More varieties with broad-spectrum resistance were identified. The most resistant varieties identified from those two groups of entries in the IUBN are listed in Table I. Very useful donor parents have been identified through the nurseries. The composition of the nurseries was recently modified to include improved breeding lines from various breeding programs. c. Genetics of Resistance. Genetic studies on blast resistance were first reported by Sasaki (1922). Takahashi (1965) reviewed the work done up to about 1963. Not much reliance can be placed on the studies because few used pure fungus strains of known pathogenicity. Systematic studies using pure cultures of known pathogenicity were begun by Niizeki (1960) and continued by Kiyosawa and co-workers. The studies were reviewed by Kiyosawa (1972, 1974). With the use of seven fungus strains of

269

DISEASE AND INSECT RESISTANCE IN RICE TABLE 1 Blast-Resistant Varieties Selected from International Blast Nurseries'

Variety

Group rb Tetep Nang Chet Cuc C46-15 Tadukan Trang Cut L. 11 Pah Leuad 111 H-5 R-6 7 (217787 Mekeo White Ram Tulasi (Sel) D25-4 M-302 Padang Trengganu 22 Ta-poo-cho-z Group rrC C46-15 Mamoriaka Carreon Huan-sen-goo Dissi Hatif Ram Tulasi Ram Tulasi (Sel) Thava Lakkanan PTB 9 Macan Tag0 Ahmee Puthe Ca 435/b/5/1 DNJ-60 Susceptible varieties Kung-shan-wu-shen-ken Fanny

Origin

Total tests 1964-1973

Susceptibility index

Resistant frequency (%)

Vietnam Vietnam Burma Philippines Vietnam Thailand Sri Lanka Senegal U.S.A. New Guinea India Burma Sri Lanka Malaysia China

302 292 307 309 263 258 3 14 291 278 276 297 292 310 239 277

1.24 1.64 1.56 1.50 1.70 1.57 1.71 1.85 1.83 1.94 1.70 1.73 1.86 1.93 1.61

98.0 88.3 93.8 94.5 94.3 94.3 92.1 92.4 91.7 92.8 91.9 93.6 90.3 87.4 91.3

Burma Malagasy Philippines China Senegal India India India Philippines Burma Indonesia Bangladesh

229 227 227 216 223 211 194 222 155 136 205 224

1.5 1 1.48 1.38 1.35 1.51 1.41 1.42 1.52 1.75 1.49 1.56 1.93

97.3 97.8 97.4 96.3 97.3 91.2 97.3 96.9 95.5 97.1 97.1 93.8

China France

246 252

4.30 4.39

24.4 19.4

aFrom Ou et al. (1975). bCroup I consists of 258 varieties selected at random and tested in IBN since 1963. 'Group 11 consists of 321 varieties selected from more than 8200 varieties after repeated tests at IRRI and was entered in IBN since 1965.

varying pathogenicity, the genetic constitution, for blast resistance of several domestic and introduced rice varieties was analyzed. Ten Ioci are designated: (1) Pi-a, (2) Pi-b, ( 3 ) Pi-J (4) Pi-i, ( 5 ) Pi-k, ( 6 ) Pi-m, (7) Pi-s, ( 8 ) Pi-t, (9) Pi-tu, and (10) Pi-z. Some are characterized by multiple allelic series. The Pi-k locus, originally identified by Yamasaki and Kiyosawa (1966) in the variety Kanto 5 1,

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GURDEV S. KHUSH

has at least three other distinct alleles-Pi-ks (Kiyosawa, 1969a), P i - k p (Kiyosawa, 1969b), and R-kh (Kiyosawa and Murty, 1969). Similarly, Pi-ta and Pi-ta2 are two distinct alleles at the Pi-fa locus (Kiyosawa, 1966, 1967b, 1969b), and R - z f are distinct alleles at the Pi-z locus (Kiyosawa, 1967a; Kukoo and Kiyosawa, 1970). The distribution of the resistance genes in different rice varieties is shown in Table 11. TABLE I1 Genes for Resistance to Japanese Isolates of Blast Fungus Identified to Date and Their Distribution in Different Varieties Gene locus

Allele

Type variety

Other varieties

Pi-a

Pi-a

Aichi Asahi

Akage, Akebono, Akibare, Kinmaze, Norin 17, Norin 18, Norin 21, Takara Towada, Jae Keun, Pal tal, Usen, Toto, Blue bonnet, Zenith, Hokushi Tami, Dawn

Pi-b

Pi-b

BL8

Tjina, Tjahaja, Bengawan, Milek Kuning

Pi-f

Pi-f

Stl

Chugoku 31

Pi-i

Pi-i

Ishikare-shiroke

Asashio, Fujisaka 5, Fukuyuki, Kitaminori, Yoneshiro, Akishinomochi, Kohi, Miyoshi, Noruho, Shinsetsu, Doazi chall, Dawn

Pi-k

Pi-k

Kanto 51

Pi-kh Pi-k p

K3 K2 Shin 2

Koshi-minori, Kusabue, Matsumae, Senshuraku, Tchi-honami, Yachiho, Dewa-no-mochi, Teine, Yuukara, Sakaki-mochi, Hakkai 219, Sanpuku Kongo, Suzukaza, Yakei-Ko, Reishiko, To-to, Choko-To, Dawn HR22 Pusur To-to, Taihung 65, Caloro, Lacrosse, Sha-tiao-tsao, Ishikarishiroke

Pi@ Pi-rn

Pi-m

Pts

Pi-s

65A15

Pi-t

Pi-t

KS 9

BL10, Tjina

Pi-to

Pi-ta

Yashiro-mochi

Pi-ta2

Pi 4

Pai-kan-tao, Tadukan, Taso-senbon, Shimokita, Pi 1 , Pi 2 Akiji, Asa-hikari, Pi 3, Satominori

Pi-z

Fukunishiki Toride I

Pi-z

Pi-zf

Tsuyu-ake, Hokushi Tami, Minehikari

Zenith, Ohy 244, 54C68, Fukei 67 C025, TKM1, C04, Morak Seplai, Kontor, Leuang Tawang 77-1 2-5, Chao Leuang 11, Toride 2

DISEASE AND INSECT RESISTANCE IN RICE

27 1

Some genes have been assigned to the respective linkage groups by appropriate linkage analysis. Thus, Pi-z and Pi-i have been assigned to linkage group I (Fukuyamz e t al., 1970; Yukoo and Fujimaki, 1970); Pi-m, Pi-k and Pi-A to linkage group VIII (Toriyama et al., 1968b; Kiyosawa, 1968); Pi-ta, to linkage group VII (Kiyosawa, 1970; Fukuyama et al., 1970); and Pi-s, t o linkage group X (Iwata and Omura, 1971). Some varieties have more than one gene for resistance. The welI-known Zenith from the United States has Pi-z and Pi-i, and Dawn has Pi-a, Pi-k, and Pi-i. The Chinese variety Hokushi Tami has Pi-a, Pi-k, and Pi-m. Several Japanese varieties-Kiho, Miyoshi, Naruho, and Shinsetsu-have the combination Pi-a and Pi-i. The distribution of resistance genes is cosmopolitan. Pi-a is present in varieties from Japan, Korea, China, India, Pakistan, and the United States; Pi-i in varieties from Japan, Korea, and the United States; Pi-k in varieties from China;Pi-ks in varieties from Japan, China, and the United States; Pi-kp in varieties from Pakistan; Pi-kh in varieties from 1ndia;Pi-ta in varieties from the Philippines and ChinaPi-ta2 in varieties from the Philippines; Pi-z in varieties from the United States; Pi-z* in varieties from India, Thailand, and Malaysia; Pi-b, in varieties from Indonesia and Malaysia; Pi-t, in varieties from Indonesia; and Pi-m, in varieties from China (Kiyosawa, 1974). Outside of Japan, two critical studies on the genetics of blast resistance have been reported. Pure cultures of known races of blast were employed in both studies. Atkins and Johnston (1965) reported a single dominant gene in Northrose and Nato that carried resistance to the United States race 1 of blast; they designated the gene as Pi 1. Zenith and Gulfrose have another dominant gene that governs resistance to the United States race 6. That gene was designated Pi 6. Pi 1 and Pi 6 showed independent segregation. Hsieh et al. (1967) identified three dominant genes for resistance in japonica strains. The genes which carried resistance to races 4, 22, and 25 from Taiwan, were designated Pi 4, Pi 22, and Pi 25. Genes for blast resistance identified in Japan, Taiwan, and the United States have not been related t o each other. Internationally coordinated genetic studies on blast are badly needed t o identify genes for resistance t o races of blast prevalent in tropical Asia, Africa, and Latin America. Resistance genes so identified would be employed in international breeding programs. d. Breeding for Resistance. Breeding for blast resistance has been in progress in different countries for at least 40 years. The work done up to 1963 in Japan, the United States, India, Taiwan, and Thailand was reviewed by Ito (1965), Atkins et al. (1965), Padmanabhan (1965a), Chang et al. (1965), and Dasananda (1965), respectively. More recent reviews are those by Ou and Jennings (1969), Ou (1972), and Toriyama (1972). To develop blast-resistant varieties, Japanese breeders have incorporated resistance genes from (1) native Japanese lowland varieties, (2) Japanese upland varieties, (3) Chinese varieties of japonica type, and (4) introduced indica vari-

272

GURDEV S. KHUSH

eties. Blast-resistant varieties Norin 6 and Norin 8 were developed in 1935 and in 1936 from the crosses of lowland Japanese varieties Joshu/Senichi and Ginbozu/ Asahi, respectively. Hybridization of the two produced Norin 22 and Norin 23. These two varieties and Yamabiko were recommended for southwestern Japan. Blast-resistant varieties Riku 132 and Fujiminori were developed for northeastern Japan, and Ishikari-shiroke for northern Japan. Yamabiko and Fujiminori possess the Pi-u gene for resistance (Ezuka et al., 1969). Ishikari-shiroke has Pi-i which gives moderate resistance to the Japanese races of blast and is still effective. The development of races virulent to Pi-u has made the gene ineffective. However, varieties with Pi-a continue to show some resistance because of the presence of polygenes for resistance (Toriyama, 1972). Several outstanding varieties-Wakaba, Wase-wakaba, Koganenishilu, Ukonnishiki, and Homare-nishiki-with resistance genes from Japanese upland varieties were developed. They were widely planted in the mountainous regions of southwestern Japan. These varieties have moderately high levels of resistance, which appears to be stable (Ujihara, 1960). They are now widely used as sources of resistance in breeding programs in Japan (Toriyama, 1972). Two Chinese varieties of the japonica type-Reishiko and To-to-that were found highly resistant to blast in Japan (Matsuo, 1952a) were used in the breeding programs. Kanto 51 and Kanto 55, two breeding lines with a high level of blast resistance, were developed and employed in the hybridization programs to produce several blast-resistant varieties, such as Kusabue, Yuukara, and Senshuraku. The varieties were very widely planted. However, they became susceptible 3 to 5 years after their release. Their resistance was conditioned by the Pi-k gene, and damage to them was due to the development of races virulent to the Pi-k gene (Matsumoto et ul., 1965). Another Chinese variety, Hokushitahmi, was also used as a source of resistance in the breeding program. Kongo and Minehikari, highly resistant progenies with the resistance gene Pi-m in addition to Pi-k (Kiyosawa, 1968) were produced. Resistance genes Pi-ta and Pi-ta2 from the Philippine variety Tadukan (indica type) were transferred to varieties of a japonica background by backcrossing (Shigemura and Kitamura, 1954; Kitamura, 1962). Japonica type lines Pi 1 to Pi 5, with high level of resistance to blast were developed. Pi 1 and Pi 2 were found to have Pi-ta (Kiyosawa, 1966), whereas the resistance of Pi 3, Pi 4, and Pi 5 was found to be due to Pi-tu2 (Kiyosawa, 1967b). With the Pi lines as parents, resistant varieties Satominori, Akiji, Shimokita, and Tosasenbon were developed and released for cultivation (Kirya e l ul., 1966; Matsuzawa et al., 1968; Toriyama et d., 1968~).Those varieties which possess resistance genes from introduced indica Varieties have been widely grown without a breakdown of resistance (Toriyama, 1965, 1972). Resistance gene Pi-z of Zenith has also been transferred to varieties with a japonica background. However, the variety Fukunishiki, which carries Pi-3 (Kiyosawa, 1967a), started to show blast susceptibility after a few years (Hirano et al., 1967). Recently, stronger genes for resistance

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from indica varieties, such as Pi-zf from TKM 1 and CO 25 (Nagai et al., 1970), and Pi-b and Pi-t from Tjina, Tjahaja, and Milek Kuning (Fujimaki and Yokoo, 1971) have been transferred to Japanese varieties. Japanese scientists distinguish between “true” resistance (caused by specific genes to races of known pathogenicity) and “field” resistance. In blast nursery tests, varieties having the same true resistance genes sometimes show differential reactions. The differences are attributed to the differences in field resistance of the varieties (Hirano et al., 1967; Asaga and Yoshimura, 1969; Hirano and Matsumoto, 1971). If varieties lack field resistance, they are severely affected by the fungus races virulent to true resistance genes. Field resistance is estimated by growing varieties in areas where races virulent t o the true resistance genes that the varieties possess are prevalent. Field resistance is also evaluated by spray inoculation with various fungus strains. Varieties that show few and small lesions when tested by the spray inoculation method are considered to have field resistance (Niizeki, 1967). Variety St 1 gave susceptible reactions against seven fungus strains when tested by the injection method of inoculation. However, it showed high field resistance when tested by the spray inoculation method (Sakurai and Toriyama, 1967). Genetic analysis showed that the high field resistance of St 1 was controlled by a single major gene, Pi-J which is linked to Pi-k with a recombination value of 20% (Toriyama et al., 1968a). The field resistance of St 1 also broke down a few years after it was bred (Yunoki et al., 1970). Thus, field resistance, as defined by Japanese scientists, is specific, controlled by major genes, and may not differ in longevity as compared with true resistance. It differs from horizontal resistance or field tolerance, which is conditioned by the polygenes and is considered lasting. Work on breeding for blast resistance in India was reviewed by Padmanabhan (1965a). Rapid progress has been made in incorporating blast resistance into improved materials at the Central Rice Research Institute (CRRI) at Cuttack; at the All India Coordinated Rice Improvement Project (AICRIP) at Hyderabad; and at other rice-breeding stations in the country. Local resistant donors (ARC 1250, MNP 36, Mahoharsali, MTU 5, BAM 7, BJ 1, TKM 6) and introduced donors (Nahng Mon S4, Sigadis, Carreon, Tadukan, Tetep, Dissihatiff, and Zenith) have been used as sources of resistance. Multilocation testing for blast of the breeding materials in nationwide coordinated trials has facilitated the work in resistance and identification of improved plant-type breeding lines with broad-spectrum blast resistance. The breeding program for blast resistance at IRRI has emphasized the use of diverse sources of resistance identified in the IUBN. Tall donor parents are crossed with improved plant-type varieties or breeding lines, and many better plant-type lines are selected from the crosses. The lines are continuously screened in the blast nurseries, two or three times a year for several years. Lines that show resistance in all the tests are again crossed with improved-plant-type

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lines having resistance to other diseases and insects, or some desirable agronomic or grain quality characteristic. They are again rigorously screened for blast resistance. In this way, many resistant lines from several resistant donors, such as H105,Nahng Mon S-4, Dawn, B589A4, Kam Bau Ngan, Cam Pai 15, Sigadis, and Tetep, have been developed (Table 111). Recently the composition of the IUBN was modified to include breeding lines in addition to donor parents. That will facilitate the early identification of lines with a broad spectrum of blast resistance and their dissemination internationally. In addition to rigorous screening in the blast nursery, all breeding materials are carefully watched for the occurrence and incidence of leaf blast and neck rot in various breeding nurseries. Those that show susceptibility are discarded. Thus, any breeding lines which reach the varietal stage must be screened at least 15 to 20 times in blast nurseries in as many seasons, and observed in field nurseries for 3 to 5 years at several locations. Highly susceptible combinations that are encountered are discarded in the early stages. As a result of continuous screenTABLE 111 Some Improved Plant-Type, Blast-Resistant Selections Developed at IRRI from Various Donor Varieties Donor parent H105

Cross

Selection IR4-93

Hl05lDgwg

Nahng Mon S-4 IR480-5-9

Nahng Mon S-4*/TN1

Dawn

IR759-54-2-2 IR1909-1-3-3

IR8/Peta3//Dawn IR84 /Dawn

B589A4

IR790-28-1-6

/TNl Peta4 /TN 1/4/1R8///H105/Dg~g//B589A4~

Kam Bau Ngan

IR1360-85-2-3

IR8/Kam Bau Ngan

Gam Pai 15

IR833-6-2-1 IR206 1-213-2-17(IR34)

Peta3/TN1//Gam Pai 15 Peta3 /TNl//Gam Pai 15/4/IR8/Tadukan// TKM62/TN1///IR24410.nivara

Tetep

IR1416-128-1-2 IR1416-131-3-6 I R 1544-2 38-2-3 IR 1544-340-6-1 IRl820-52-2-4 IR2035-290-2-3

Peta4/TNl//Tetep Peta4 /TN 1//Tetep IR24/Tetep IR24/Tetep IR24//Mudgo/IR8///Peta4 /TN1 //Tetep

IR1529-680-3

Sigadis' /TN 1//IR24

Sigadis

Peta4/TN1//Tetep///Peta3/TN1//HR21/4/ IR24//M~dgo/IR8///IR24~/0. nivara

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ing, the advanced breeding lines at IRRI have good levels of resistance. Of the eleven IRRI named varieties three (IR28, IR29, and IR34) have strong blast resistance, inherited from the common parent Cam Pai 15. High levels of resistance are not involved in the parentage of the remaining eight varieties, but some (IR20, IR26, and IR32) show moderately resistant reactions in the blast nursery. Although IR8 and IR5 have been classified susceptible in the blast nursery, they have rarely been infected with blast under lowland conditions in Asia where they were widely grown. It appears that those varieties have adequate levels of field tolerance that are not detected in the blast nursery. Serious yield losses from blast in lowland rice in tropical Asia have rarely been reported in recent years. Apparently, the present varieties have adequate resistance or field tolerance. However, what happened in Japan earlier could occur in tropical Asia. As the average yields per unit area increase with increased use of fertilizers and other inputs, blast might become a limiting factor to higher production. A coordinated international approach t o the development of blastresistant varieties needs to be adopted. It should include: (1) identification of most widely distributed blast races in the cooperating countries, (2) identification of blast resistance genes through genetic analysis using the identified races, (3) incorporation of distinct resistance genes into isogenic lines using varieties with field tolerance as recurrent parents, and (4) programmed release of these lines, either successively when the previous one becomes susceptible or as multilineal varieties. Several genes for resistance should also be combined in the same improved variety.

2. Sheath Blight Sheath blight is perhaps the second most important fungal disease of rice. It ranks second only t o blast in the yield losses it causes. The fungus causing the disease has been called Corticium sasakii (Shirai) Matsumoto or Rhizoctonia soZani Kuhn, and several other names. Earlier it was thought that the disease occurred only in Asia, but recently it has been reported in Brazil, Surinam, Venezuela, and Madagascar (Ou, 1972). Our observations and reports from our collaborators in other countries indicate that the attacks of sheath blight have greatly increased in tropical Asia in recent years. The increased incidence is attributed to the greater use of highyielding, high-tillering, short-statured, and early-maturing varieties as well as to higher plant populations and greater use of nitrogenous fertilizers. The disease incidence is likely to increase even further with the widespread use of high-yielding varieties and better management practices. a. Variution in Pathogenicity. Differences in the pathogenicity of fungal isolates were reported by Chien and Chung (1963). They classified 300 isolates into seven culture types and six physiological races based on the disease re-

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actions of 16 varieties. The susceptible and resistant reactions used in separating the races were not so clear-cut, however. Differences in pathogenicity of isolates were also noted by Akai et al. (1960), Tu (1967), and by IRRI pathologists (IRRI, 1974). Parmeter (1970) also reported that the fungus isolates differ in virulence and host range. b. Varietal Resistance. IRRI pathologists have tried various screening techniques to determine differences in varietal resistance-seedling-stage inoculation, detached flag-leaf inoculation, leaf-sheath inoculation, and adult-plant inoculation. Distinct varietal differences were noted with each method of inoculation, but the results did not completely agree. Some varieties showed resistance reactions with one method, susceptible reactions with another. Some inconsistencies in reaction between the seedling stage and the adult plant stage can be explained by the relation between plant age and disease development. Plants at the flowering stage are more susceptible than those at the seedling or tillering stages. Since the disease is more important after the flowering stage, the adult stage inoculation method has been adopted for screening materials at IRRI (IRRI, 1974). The reactions of rice varieties to sheath blight range from highly susceptible to moderately resistant. Resistant or highly resistant varieties have not yet been found. Most moderately resistant varieties that have been identified show a disease index of about 30; the susceptible varieties show a disease index of 80-90. The disease index is calculated as the percentage of infected leaf sheath area from a 10- to 25-tiller sample. From a sample of over 1000 varieties and breeding lines screened by plant pathologists at IRRI, several moderately resistant entries have been identified: Kataktara Da-2, Ta-poo-cho-z, Charnock, F’tbl8, Carreon, Bahagia, Colombia 1, CS2, OS4, Mehran, Sinaloa A68. Several improved plant-type breeding lines such as IR442-2-58, IR533-1-89, IR1330-3-2, IR1360-87-1, and IR1093-148-3 have good levels of resistance. On the basis of field tests in Japan, Hashioka (1951a) reported that varieties from India, Thailand, Burma, and Europe were more resistant than local varieties. Varietal differences in reaction were noted by Hseihetal. (1965) in Taiwan. Screening done in India showed CR1-6, CR44-11, Saket 1, and Sona as moderately resistant (H. K. Saksena, personal communication). c. Inheritance of Resistance. Hashioka (1951 b) reported that resistance to sheath blight is dominant over susceptibility. The F2 populations from crosses between resistant and susceptible parents were either mostly resistant or segregated in a ratio of 3 resistant to 1 susceptible. d. Breeding for Resistance. Work on breeding for resistance to sheath blight has not been done anywhere, not even in Japan where the disease has been a serious problem for many years. The unavailability of donor parents with usable levels of resistance has been the main cause of lack of progress in that area

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(Toriyama, 1972). Faced with the ever-increasing threat of the disease in tropical Asia and requests for resistant germ plasm, the Genetic Evaluation and Utilization (GEU) program at IRRI expanded its efforts to screen and develop germ plasm with levels of resistance higher than those available at present. The work involves: 1. Continuous evaluation of germ plasm. To date, less than 3% of the entries of available germ plasm have been screened. It is conceivable that varieties with higher levels of resistance will be found if enough materials are screened. 2. Thorough screening of all the advanced breeding lines and elimination of all highly susceptible materials. To raise the frequency of resistance genes in our improved materials, parents with high susceptibility are not entered in the crossing blocks. 3. An organized breeding program, using selected lines with moderate levels of resistance. The program aims at pyramiding minor genes for resistance from several parents into the same line by a method of recurrent selection. The method is discussed in another section of this chapter. 3. BrownSpot Brown spot disease, caused by Cochliobolus miyabeanus (Ito et Kuribayashi) Drechsler ex Dastur, is more commonly known by its other scientific name Helminthosporium olyzae Breda de Haan. The disease has been known for the last 75 years. It caused the most devastating rice yield losses that led to the Bengal famine of 1943 (Padmanabhan, 1973). Little work has been done to develop varietal resistance to the disease, perhaps because plant pathologists generally believe that it does not cause serious damage. Serious damage is generally associated with other primary problems such as nutrition and physiological disorders. Baba (1958) and Takahashi (1967) found that the disease was associated with the physiological disorder called aikiochi and that the symptoms were hard to find on plants growing on normal soils. The aikiochi disorder occurs mainly on peat, or on sandy or muck soils. Outbreaks of the disease in Sri Lanka in the province of Uva in 1943, and in the district of Polonnaruwa in 1952 occurred on the rice crop grown on problem soils (Abeygunawardena, 1967). Kanjanasoon and Sitthichai (1967) reported that the plants infected with yellow orange leaf (tungro) virus appeared more susceptible to Helminthosporium disease during the virus epidemic of 1966 in Thailand. Recent outbreaks of Helminthosporium in the Ngale area of Indonesia and Bohol island in the Philippines occurred on potash-deficient soils (S. H. Ou, personal communication). a. Variation in Pathogenicity. Not much critical information on the variation in pathogenicity of the fungus is available. From the reaction of 132

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monosporic strains on 15 rice varieties, Tochinai and Sakamoto (1937) found a wide range of pathogenicity-from very weak to extremely virulent. Variation in pathogenicity of fungus isolates was also noted by Nawaz and Kausar (1962), Misra and Chatterjee (1963), and Vorrauri and Giatgong (1970). No variation in pathogenicity was encountered by Padmanabhan (1953). b. Varietal Resistance. The disease epidemic of 1942 in Bengal generated great interest in India in identifying the resistant germ plasm. Ganguly (1946) reported that varieties Dakar Nagar 273-32, Patnai 549-33, Kalma 219, and Na'gra 41-14 were resistant in Bengal. Ganguly and Padmanabhan (1959) and Padmanabhan et al. (1966) screened 490 varieties and found Ch13, Ch41, Ch45, T498-2A, C020, Bam 10, T998m, T2112, T2118, and T960 resistant. Five varieties-Taichung Native 1, Muey Nawng 62M, Leuang 34, Leuang Thong 82, and a hybrid line PJP54 X IRH8-BKN56-7-106-were found resistant in Thailand (Kanjanasoon and Sitthichai, 1967). Hainan No. 217 and Chiu Tiu Chiu were also found resistant (Asada et al., 1954). c. Inheritance of Resistance. Information on the genetics of resistance is fragmentary. Nagai and Hara (1930) reported that resistance in a Korean strain of rice was controlled by a single dominant gene. On the other hand, Adair (1941) found that resistance behaved like a recessive character. d. Breeding for Resistance. In the United States, C19515 was found to be highly resistant to Helminthosporium It was crossed with Texas Patna and TP4TP4-9. Selections from the crosses were further crossed with Century Patna 23 1 and Bluebonnet. Several resistant progenies were selected from these crosses but had low yield potential. In 1966, the blast- and brown spot-resistant variety Dawn was released in the United States (Ou, 1972). At present the author is not aware of any active program on breeding for resistance to brown spot. However, advanced generation breeding lines are thoroughly evaluated for their reaction to the disease in India at CRRI and AICRIP.

4. Narrow Brown Leaf Spot Narrow, brown leaf spot caused by Cercospora oryzae Miyake is recorded in all rice-growing countries of Asia, Africa, North America, Central America, and Australia. The disease produces short, linear, brown lesions commonly on leaves. The lesions generally appear in large numbers during the later stages of growth. The disease is considered of minor importance in most countries. However, it attracted much attention in the United States during the 1930s and 1940s. The disease appeared in epidemic proportions because some important commercial varieties (Blue Rose for example) of that time were highly susceptible. A similar situation developed in the Philippines in 1972-1973 when IR20 was widely grown in the country. IR20 is highly susceptible to Cercospora, and the

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fields planted to it were readily identified by the reddish brown appearance of the leaves caused by heavy infection. a. Variation in Pathogenicity. Using a set of 8 differential varieties, American workers identified several races of the fungus. Eight races were identified by Ryker (1943, 1947), and races 9 and 10 were reported by Ryker and Cowart (1 948). b. Varietal Resistance. Germ plasm was systematically screened in the United States by Tullis (1937), Ryker and Jodon (1940), and Adair and Cralley (1950). Several highly resistant varieties were identified. Outstanding among them were Rexoro, Fortuna, Nira, Iola, (2.1.461, C.I.440, Asahi, and Kamrose. During the severe disease incidence in 1972-1973 in the Philippines, breeding nurseries of IRRI were badly attacked by narrow brown leaf spot. Most of the IRRI parental materials and advanced generation breeding lines were classified according to their reaction to narrow brown leaf spot. Varieties IR24, IR26, IR28, IR29, and IR34 and several breeding lines were highly resistant. c. Inheritance of Resistance. The inheritance of resistance was investigated in the United States. In six crosses between resistant and susceptible parents studied by Ryker and Jodon (1940), F2 populations segregated in a ratio of 3 resistant to 1 susceptible, thereby showing monogenic dominant control of resistance. Two linked genes for resistance, one giving resistance to race 1 and the other to race 2, were reported by Ryker and Chilton (1942). Varieties susceptible to race 1 were resistant to race 2, and vice versa. Segregating populations from crosses between the two parents yielded only a few recombinants (plants with resistance to both races). A large number of crosses between resistant and susceptible parents were investigated by Jodon et al. (1944). In 35 cross combinations, single gene segregation was obtained, in 13 crosses two genes segregated. d. Breeding for Resistance. In the United States, special attention was devoted to selecting varieties with resistance to narrow brown leaf spot during the 1930s and 1940s. Varieties Delrex, Sunbonnet, Toro, and Selection 44C507, bred during that period, were resistant to the disease (Jodon, 1953, 1954, 1955). Owing to the insignificance of the disease, no rice-breeding program in the world is working on it. However, the reaction of all future varieties to it should be determined to avoid the release of materials with extra susceptibility. Otherwise, the minor disease could become a major disease.

B. BACTERIAL DISEASES

Several bacterial diseases of rice have been identified. Some, such as bacterial stripe, bacterial sheath rot, and black rot, are of minor importance. The available

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GURDEV S. KHUSH

information on these diseases is reviewed by Ou (1972). Bacterial blight and bacterial streak are of international importance and cause considerable yield losses. 1. Bacterial Blight

Bacterial blight, one of the most serious diseases of rice, occurs in all rice-growing countries of Asia and in Australia, but not in Europe, Africa, or North and South America. The causal bacterium is commonly referred to as Xanthornonas oryzae (Uyeda and Ishiyama) Dowson. The disease symptoms usually appear at the flowering stage in the field. Lesions start at the margins of the leaf blades; they enlarge and form a wavy margin. As the disease advances, the lesions cover the entire leaf blade and may even advance into the leaf sheath. Under heavy disease pressure all the leaves may be killed, and severe yield losses may result. In tropical areas, the attack may occur at the seedling stage; some tillers or even the entire plant may die. When the attack occurs at the seedling stage, the disease is referred to as Kresek. a. Variation in Pathogenicity. Pathogenic variability of the organism was suspected in 1957 in Japan when a resistant variety Asakaze was severely attacked by bacterial blight. lnvestigators attempted to classify the bacterial strains on the basis of virulence. Using the reactions of differential varieties, Washio et al. (1966) and Sakaguchi et al. (1968) classified the bacterial strains into three groups (I, 11, and 111) and the varieties into four groups. The varieties of the Kinmaze group are susceptible to all bacterial groups. The varieties of the Kogyoku or Kidama group are resistant to the bacterial isolates of group I, but susceptible to the isolates of groups I1 and 111. The Rantaj-emas group varieties are resistant to the isolates of groups I and 11, but susceptible to the isolates of group 111. The varieties belonging to the Wase-Aikoku 3 group are resistant to the isolates of the three groups (Table IV). Tin Win (1974) identified a new Philippine strain the so-called Isabella strain to which resistant varieties with a dominant gene for resistance, such as IR20, are moderately susceptible. Differences in virulence patterns of 161 isolates from 37 locations in India were noted by Kauffman and Rao (1972). b. Varietal Resistance. Thorough screening of germ plasm was first done in Japan. Most Japanese varieties belong to the Kinmaze group and are susceptible to all Japanese isolates of the bacterium. A few varieties (particularly those bred for resistance after 1935) and some introduced varieties (Bomba and Gangsale Bhatta) belong to the Kidama group (Table IV). Most introduced resistant varieties of the indica type belong to the Rantaj-emas group. No Japanese variety belonging to this group has been identified. A small number of varieties-Wase Aikoku 3, Nakashin 120, TKM6, and Lead rice-belong to the Wase Aikoku group (Washio et al., 1966; Sakaguchi et al., 1968; Ezuka and Horino, 1974).

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DISEASE AND INSECT RESISTANCE IN RICE

TABLE IV Differential Reactions between Rice Varieties and Strains of Bacterial Leaf Blight in Japan Reaction to bacterial group Varietal group

Representative varieties

I

I1

I11

Kinmaze group

Kinmaze, Jukkoku, Norin 37, Shimotsuki, Asahi, Originario

S

S

S

Kogyoku group

Kogyoku, Zensho 26, Norin 27, Asakaze, Hayatomo, Koganemaru, Kokumasari, Nishikaze, Kanto 60, Boma, Gangasale Bhatta

R

S

S

Rantaj-emas group

Rantaj-emas, Tadukan, Tetep, Nigeria 5 , Basilanon, Kele, Pinulupot

R

R

S

Wase Aikoku group

Wase Aikoku 3, Nakashin 120, TKM6, Lead Rice

R

R

R

Thousands of varieties maintained in the germ plasm bank at IRRI have been evaluated for resistance to bacterial blight by IRRI plant pathologists (IRRI, 1966, 1967a, 1973, 1974; Ou et aL, 1971a; Kauffman e t al., 1973). As a result, more than 300 varieties are known to be resistant to the Philippine isolates of the bacterium. Some are resistant to the Isabella strain-a new strain from the northern part of the Philippines-as well as to the IRRI strain, which is representative of Philippine strains in general. Varieties BJ1 and Dular from India, and AZ192 and Hashikalmi from Bangladesh are resistant to both strains. IR20, IR22, and breeding lines derived from Sigadis are susceptible to the Isabella isolate, but resistant to the IRRI isolate (IRRI, 1974). Most resistant germ plasm identified at IRRI comes from three geographical regions. A large number of varieties come from Bangladesh, Nepal, and West Bengal and Assam states of northeast India. The area may be designated as gene center 1 for bacterial blight. Another group of resistant varieties comes from south India and Sri Lanka. That region is designated as gene center 2. The third gene center is Java and the adjoining islands in Indonesia. A large number of resistant varieties belong to this region (Fig. 1). A very small number of resistant varieties (two or three each) come from Malaysia, the Philippines, Taiwan, Vietnam, Laos, and Thailand. Very ambitious programs to screen the native germ plasm for resistance to local isolates of the bacterium are under way in India, Bangladesh, Burma,

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GURDEV S. KHUSH

FIG. 1. Distribution of bacterial blight and bacterial streak in Asia. The three gene centers for bacterial blight resistance are also shown.

Thailand, and Indonesia. A large number of resistant varieties have been identified at CRRI (S. Y. Padmanabhan, personal communication). c. Inheritance of Resistance. Inheritance of resistance to Japanese isolates of the bacterium was investigated by Nishimura and Sakaguchi (1959), Sakaguchi (1967), Sakaguchietal. (1968), Murata(1967), andEzukaetal. (1970,197s). The resistance of the Kidama group to isolates of group I of the bacterium is controlled by a single dominant gene designated Xul. The varieties of the Rantaj-emas group have two genes for resistance, Xal and Xa2. Xa2 conveys resistance to the bacterial isolates of group 11. X a l and Xa2 are linked with a crossover value of 3% and have been located on chromosome 11 (Nishimura, 1961). No variety with Xu2 alone has been identified, although segregates with Xa2 Xu2 genotype have been obtained in the segregating populations of crosses between varieties of the Rantaj-emas group and the Kmmaze group. Segregates with Xal Xal genotype have also been obtained from varieties of the Rantajemas group. Variety Pi1 (derived from Tadukan of the Rantaj-emas group) shows resistance against the pathogens of group I, but not against those of group 11, thereby indicating that it inherited only Xal from Tadukan (Toriyama, 1972). The nature of resistance in Wase Aikoku 3, which is resistant to all three groups of bacterial isolates, is somewhat different. Whereas varieties having Xal

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or Xa2 show resistance at all stages of growth, Wase Aikoku 3 is susceptible if inoculated at the seedling stage, but resistant if inoculated at the adult plant stage. Resistance in this variety is controlled by a single dominant gene first designated as Xa-w (Ezuka et al., 1970, 1975). However, in order to conform to the international rules of gene symbolization, Petpisit et al. (1977) changed the designation of this gene to Xa3. Xa3 segregates independently of X a l and Xa2. It should be possible to combine Xa3 with either X a l or Xa2, or both. Several studies on the inheritance of resistance to Philippine isolates of the bacterium carried out at IRRI (Heu et al., 1968; Murty and Khush, 1972; Murty e t al., 1973; Librojo et al., 1976; Petpisit et al., 1977; Olufowote et aZ., 1977) revealed the existence of three loci for resistance. Varieties Sigadis, Pelita I/1, and Semora Mangga from Indonesia, TKM6 and MTUl5 from India, Hom Thong from Laos, and IR22 possess single dominant genes for resistance which are allelic t o each other. This locus has been designated as Xa4. Unlike other varieties with Xa4 for resistance, Semora Mangga is susceptible at the seedling stage but resistant at the booting stage or just before flowering. The gene for resistance in this variety was designated Xa@ to distinguish it from Xa4a in IR22. Varieties BJ1, DZ192, Dular, Kele, Chinsurah Boro 11, and Hashikalmi each possess a single recessive gene for resistance. These genes are allelic to each other. The locus with recessive genes has been designated xa5; Xa4 and xa.5 segregate independently of each other. Varieties Zenith and B589A4 from the United States, and Malagkit Sungsong from the Philippines have allelic genes for resistance at the third locus. Thls locus is closely linked with Xu4 but, as expected, is independent of xa5. Varieties belonging to the Zenith group are susceptible at the seedling stage but resistant at booting and adult plant stages. The F1 plants of Zenith and susceptible parents such as T N l , are susceptible at seedling and booting stages but resistant at the post-flowering stage. Thus the heterozygotes show differential reactions at different stages of growth. Therefore, if the F2 populations of these crosses are inoculated at the booting stage, resistant and susceptible plants will be obtained in a ratio of 1 :3. If inoculation is done at post-flowering stages, the ratio becomes 3: 1. The inheritance of resistance and the allelic relationships of resistance genes of more than 100 varieties are now under investigation at IRRI. The available information indicates that a majority of resistant varieties from gene center 1 have xa5 for resistance, whereas resistant varieties from gene centers 2 and 3 have Xa4 for resistance. In India, studies on inheritance of resistance to Indian isolates of the bacterium are under way at CRRI and AICRIP (Jayaraj et al., 1972; S. Devadath, personal communication). d. Breeding for Resistance. Much work on breeding for resistance to bacterial blight in Japan is reviewed by Mizukami (1966), Fuji and Okada (1967), Mizukami and Wakimoto (1969), and Toriyama (1972). Although bacterial blight is known to have caused damage to the rice crop in Japan since 1884, the

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first resistant plant was identified only in 1926. In that year, one plant resistant to bacterial blight was selected from a field of the susceptible variety Shinriki at Kagoshima Agricultural College. The single plant was the ancestor of the resistant strain Kono 35. In spite of its resistance, Kono 35 did not become a popular variety because of its inferior agronomic traits. As a source of resistance, however, it produced several resistant varieties. In 1936 Kono 35 was crossed with Asahi 1 at Kumamoto Breeding Center, and Norin 27 was bred and released in 1946. The latter variety was planted widely in western Japan, but the area planted to it soon decreased because of its susceptibility to blast. At Kyushu Agricultural Experiment Station, two resistant varieties-Asakaze and Hayatomo-were developed from the crosses Takara/Norin 27 and Norin 37/Norin 27, respectively. Asakaze was attacked by bacterial blight in 1957; the pathogen was a new strain of bacterial blight (Kuhara e l al., 1958). Hayatomo was released in 1964 and became a popular variety in the northern coastal areas of Kyushu. Nishikaze. another blight-resistant and early maturing variety, was bred at Kyushu in 1967 from the cross of Asakaze and Kinmaze. At the Aichi Prefectural Agricultural Experiment Station, two resistant varieties-Shiga-Sekitori 11 and Shobei-were used as sources of resistance. From a cross of Shiga-Sekitori 11 and Shoyu made in 1924, the breeding lines Zensho 17 and Zensho 26 were developed. Zensho 26 was crossed with Jikkoku in 1953; three varieties-namely Hoyoku in 1961, Kokumasari in 1962, and Shiranui in 1964-were developed from this cross. The other resistant variety Shobei was crossed with Shirosenbon; from this cross, Kogyoku and Taiyo were released in 1932 and 1934, respectively, Kogyoku was used as parent in many crosses; several very successful varieties, such as Sachikaze and Nihonbare, were released in 1960 and 1963, respectively. All bacterial blight-resistant varieties developed in Japan between 1930 and 1972 inherited their resistance from Kono 35, Shiga-Sekitori 11, and Shobei. Later analysis showed that all of them are resistant to pathogens of group I, but are susceptible to pathogens of groups I1 and 111, and have XaZ for resistance. Thus, although the breakdown of resistance governed by XaZ was noted in 1957 and Beniya district of Kyushu, Japanese breeders continued to release varieties having X a l for resistance. As far as the author knows,no commercial variety having Xa2 or Xa3 for resistance has been bred in Japan to date; however, Toriyama (1972) reported that the breeding lines Chugoku 45 (with resistance genes from Wase-Aikoku) and X38 and X43 (with resistance genes from “Lead rice”) have been developed. The breeding program for blight resistance at IRRI has resulted in several successful varieties and breeding Iines that are grown widely in Asia and have served as parents of numerous crosses at IRRI and in national breeding programs. IR20 and IR22 released in 1969; IR26 in 1973; IR28, IR29, IR30 in 1974; and IR32 and IR34 in 1975 are all resistant. Several IRRI breeding lines

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released in other countries-Palman 579 (IR579-48) in India, Chandina (IR5321-176) and Mala (IR272-4-1) in Bangladesh, TN73-2 (IR1561-228) in Vietnam, and IR36 and IR38 in the Philippines are also resistant. Many tall donor varieties were utilized as sources of resistance to bacterial blight in the breeding program at IRRI to insure the diversity of sources of resistance (Khush and Beachell, 1972). Improved plant-type breeding lines with bacterial blight resistance, high yield potential, good grain quality, and resistance to several other diseases and insects have been developed from crosses involving those parents (Table V). Later, genetic analysis showed that a great majority of IRRI breeding lines as well as all IRRI named varieties have the dominant gene Xu4 for resistance. However, as Table V shows, improved plant-type lines with xu5 as well as the Zenith gene for resistance exist among the IRRI breeding materials. The proportion of improved germ plasm with these genes is likely to increase as the breeding lines from the crosses made recently are selected. The resistant lines listed in Table V have been made available to scientists in other countries as sources of resistance in crossing programs. Bacterial blight resistance is one important breeding objective of almost all breeding programs in Asia. Pelita I l l , a popular high-yielding variety bred in Indonesia in 1971, is resistant. This variety and several resistant lines from IRRI are parents of many crosses made in Indonesia in recent years. The segregating progenies are being thoroughly screened. In Malaysia, none of the recommended high-yielding varieties is resistant. However, numerous breeding lines from crosses involving IR20, TKM6, Zenith, and Kaoshiung 52 as donors of resistance to bacterial blight have been developed. None o f the important recommended tall varieties in Thailand is resistant to bacterial blight. Several resistant varieties and breeding lines from IRRI have been utilized in the breeding program. Sigadis has been used extensively as a source of resistance. Many advanced breeding lines are resistant. In Bangladesh, several high-yielding varieties (IR20, Chandina, Mala, and BR4) and many advanced breeding lines are resistant. In India, o f the more than 50 high-yielding varieties recommended to date, only IR20, Ratna, and Palman 579 are resistant. Many advanced breeding lines are resistant. Tall donors BJ1, Malagkit Sungsong, Zenith, TKM6, Sigadis, and Lacrosse X Zenith-Nira, and the dwarf varieties and breeding lines, IR20, 1R22, IR26, RP5-32, and RP3 1-49, are the parents in numerous crosses. In terms of percentage, perhaps the largest area covered by bacterial blight-resistant varieties is in the Philippines and in the southern part of Vietnam. In the Philippines, 65% of the total rice area is planted to the high-yielding varieties IR20, IR26, IR28, IR30, IR32, IR34, IR36, IR38, and the experimental selection IR1561-228-3. All are resistant to bacterial blight. About 30% of the rice area in the southern part of Vietnam is under high-yielding varieties, most of it under blight-resistant IR20, IR26, IR30, and TN73-2 (IR156 1-228-3).

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GURDEV S. KHUSH

TABLE V Some Improved Plant-Type, Bacterial Blight-Resistant Varieties and Selections Developed at IRRI from Various Donor Parents

Donor uarent

Variety/ Selection

Cross

Resistance gene

IR156 1-228

Peta3 /TNl//TKM6 IR24/TKM6 Peta3 /TNl//Gam Pai 15/4/IR8/Tadukan// TKM6'/TNl///IR24* /Oryza nivara Peta3/TN 1//Cam Pai 15/4/IRS/Tadukan// TKM6' /TNl///IR244 10.nivara IR24/TKM6//IR204/O.nivara IR20' 10.nivara//CR94-13 Peta3/TNl//Gam Pai 15/4/IR8/Tadukan// TKM62/TN1///IR244 /O.nivara IR8/Tadukan//TKM6*/TN 1

Sigadis

IR1529-680-3

Sigadis' /TNl //IR24

Xa4

W1263

IR1330-3-2 IR203 1-238-5

Leuang Tawang/IR8//W1263 IR24'/0. nivara///Peta4/TNl//Tetep/4/ Leuang Tawang/IR8//W1263

Xu4 xa4

Tadukan

IR22 IR579-48

IRd/Tadukan IRllTadukan

xa4' ~a4'

DZ192

IR 1545-339

IR24/DZ192

xu5

B589A4

IR790-28-1

Peta4 /TN1/4/IR8//H10S/Dgwg///BS89A42 /TNl ?

M. Sungsong

IR944-102

TN1/Malagkit Sungsong//IR8

Ib

Zenith

IR1698-241-2

IR84 /Zenith

?b

Wase Aikoku

IR1694-3-2

IR83/Wase Aikoku

?'

TKM6

IR20 IR26 IR28 IR29 IR30 IR32 IR34

Xu4 Xu4 Xu4 Xu4 Xu4 Xu4 Xa4 Xu4

"Source of Xu4 in this cross is not known,as Tadukan is not resistant to Philippine isolates. bB589A4, Malagkit Sungsong and Zenith have the same gene for resistance but gene symbol has not been assigned as yet. 'Genetic analysis of Wase Aikoku for resistance to Philippine isolates of the bacterium has not been completed.

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2. Bacterial Streak Bacterial streak or bacterial leaf streak disease, caused by Xanthomonas translucens f. sp. orizicola (Fang et a].) Bradbury, is widely distributed in tropical Asia (Fig. 1). It has not been reported in Japan or in other temperate countries. a. Variation in Pathogenicity. Ou et al. (1971a) compared the virulence of 150 isolates of the bacterium from the Philippines against rice varieties Peta and Pah Leuad 111. Some isolates were weakly virulent, a few were highly viiulent; the majority were of intermediate virulence. Four least virulent isolates, three most virulent isolates, and one isolate of intermediate virulence were inoculated on 36 selected rice varieties with varying degrees of disease reaction. The resistant varieties were resistant to all isolates, and the susceptible varieties exhibited susceptibility to all the tested isolates. The results showed that although strains differ in virulence, pathogenic races with contradictory reactions in a given variety do not exist. Shekhawat et al. (1972), however, reportedly observed distinct race differences when they inoculated 15 isolates on seven differential varieties. 6. Varietai Resistance. Several varieties resistant t o bacterial streak have been identified from screening trials conducted at IRRI (Goto, 1965; Ou et al., 1971a) and at CRRI (Rao et al., 1972; Row et al., 1968). Varieties DZ50, Milbuen 5 , Blue Rose, Hashikalmi, and DZ192 looked most outstanding. In India, varieties Zenith, Tetep, H4, S67, and C 0 4 were resistant in the field trials. c. Inheritance of Resistance. Inheritance of resistance to bacterial streak has not been investigated, but it appeared to be quantitative in nature. d. Breeding for Resistance. Since the disease is not so serious, organized efforts to breed for resistance have not been made anywhere. At IRRI breeding materials are evaluated regularly for their reaction to bacterial streak under field conditions, particularly during the rainy season. All lines showing high susceptibility are discarded. Some improved plant-type lines such as RP291-20, IRl545-339, IR833-12, and IRI 541-76, were selected for their good levels of resistance. Those lines are parents of many crosses in our program.

C. VIRUS DISEASES

Twelve virus diseases of rice have been clearly recognized. Five occur within the borders of a single country and do only minor damage. These five are giallume (Italy), yellow mottle (Kenya), transitory yellowing (Taiwan), necrosis mosaic (Japan), and black-streaked dwarf (Japan). Because of their minor importance, n o work on breeding for resistance to them has been done.

288

GURDEV S. KHUSH

Yellow dwarf is perhaps the most widespread of the virus diseases of rice. It occurs in all rice-growing countries of tropical as well as temperate Asia. However, no serious damage from it has been reported anywhere. Only a few virus-infected plants are observed in any field at a time, perhaps because of the longevity of the incubation period of the virus in the plant as well as in the vector. The virus requires an incubation period of over a month in the vector before it is transmitted to the plant, and an incubation period of more than a month in the plant before the symptoms appear. Under these conditions, not enough plants ever become diseased to provide the inoculum for a disease outbreak. Orange leaf virus has been reported in the Philippines, Thailand, India, and Sri Lanka. It is a self-eliminating disease-the diseased plants die and the few that survive cannot provide a continuous supply of inoculum. Plants infected with orange leaf are rarer than those with yellow dwarf. The two diseases are considered of minor importance, and no work in breeding for resistance to them has been done. Five virus diseases are subjects of active breeding programs. Tungro and grassy stunt occur only in tropical Asia; stripe and dwarf occur only in temperate Asia; hoja blanca occurs in North, South, and Central America. This chapter will deal with only those five. For a thorough treatment of the subject of virus diseases of rice, the reader is referred to IRRl(1969), Ling (1972), and Ou (1972). 1. Tungro

Tungro is the most serious disease of rice. It lias been known in the Philippines since the mid-1930s under various names, such as rice dwarf or stunt disease (Reyes, 1957; Reyes et al., 1959), accep nu pula (red disease) or stunt disease (Serrano, 1957), and rice cadang cadang (Agati and De Peralta, 1939). It is transmitted by Nephotettix virescens. Its viral nature was clearly established by Rivera and Ou (1965). A similar disease called Penyalut merah or red disease has been known in Malaysia since 1938. Recent studies (Ou et al., 1965; Ou and Goh, 1966) have shown that Penyakit merah is caused by a virus that is identical to the tungro virus of the Philippines. Likewise, mentek disease of rice, known in Indonesia since 1859, is now believed to be simdar to tungro (Ou, 1965b; Rivera et aZ., 1968). A virus disease reported in Thailand and originally called yelloworange leaf (Wathanakul, 1965) is now believed to be the same as tungro. A virus disease first referred to as leaf yellowing (Raychaudhuri et al., 1967) in India is also considered identical to tungro (John, 1968, 1970; Raychaudhuri and John, 1970). The widespread occurrence of tungro in Bangladesh is also known (Nuque and Miah, 1969; Lippold et al., 1970). The virus disease referred to by various names in different countries is thought to be caused by the same virus or strains of the same virus, and the name tungro is now generally accepted and

DISEASE AND INSECT RESISTANCE IN RICE

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widely used in all those countries. The distribution of the virus is shown in Fig. 2. Although it has not been reported in Vietnam, Cambodia, Laos, Burma, and Sri Lanka, it is probably present in those countries. u. Vuriation in Strains. At IRRI two strains of tungro virus-S and M-are known to produce differential chlorotic symptoms on varieties FK-135 and Acheh (Rivera and Ou, 1967). Strains of the virus collected from different parts of India produce differential symptoms on several rice varieties (Shastry et ul., 1972b) and appear to differ in severity. Some strains produce weaker disease reactions than do others. However, the resistant varieties are more resistant to all strains, and the susceptible varieties more susceptible. More significant will be information on the strain variation among countries. However, no critical information on the subject is available. b. Varietal Resistance. Several resistant varieties have been identified through natural infection in the field or through artificial inoculation in the field or the greenhouse. In the first field screening for tungro resistance, Fajardo et al. (1964) identified the varieties BE3 and Peta as resistant and BP176, Wagwag, Raminad, Macatampal, and Mancasar as susceptible. Extensive field tests for resistance have been conducted in Thailand at Bangkhen Rice Experiment Station (Wathanakul and Weerapat, 1969), the Philippines (IRRI, 19731, Indo-

FIG. 2. Distribution of tungro, grassy stunt, and stripe and dwarf virus diseases in Asia.

290

GURDEV S. KHUSH

nesia (van Halteren and Sama, 1974), and India (A. Anjaneyulu, personal communication). The field tests identified several varieties with good levels of resistance (Table VI). The field-screening techniques consist of planting the test varieties with susceptible and resistant checks in fields where large numbers of diseased plants and insect vectors are present. A susceptible variety is planted around the borders of the test plots as a disease spreader. The tests can be conducted only during years of disease outbreak in a particular area. To overcome this uncertainty, Dr. A. Anjaneyulu at CRRI devised a procedure to create appropriate disease pressure in the test plots in the absence of the disease in the area. Seedlings of a tungro-susceptible variety are artificially inoculated in the greenhouse and planted as spreader in rows around the test plots 2 weeks before the test varieties. The planting time of the materials is arranged so that the rapid tillering phase of the plants coincides with the maximum population density of the insect vectors. The peak population of the insect vector at Cuttack occurs in September-October every year. The insects acquire the disease from the inoculated plants and spread it to the test plots. The test has been going on successfully for 4 years, and a large number of varieties and breeding lines have been evaluated. One drawback in all the field screening tests is that they do not discriminate between resistance to tungro and resistance to the vector. Under light to moderate disease and insect pressure, strong resistance to the vector alone is adequate to protect the crop from the disease. Even if susceptible to the virus, varieties with high levels of vector resistance conditioned by the dominant genes Glhl and GZh2 (see Section III,CJ,c on insect resistance) get no disease or very little. It appears that insects do very little feeding on varieties with high levels of resistance, and those varieties thus escape infection. Weak insect resistance, such as that conditioned by Glh3 or the Peta gene, does not provide much protection against the virus. Enough insect feeding occurs on the weak varieties that become inoculated and diseased. Under very heavy insect and disease pressure, resistance to the vector alone is not sufficient to protect the variety from the disease. A mass screening method by artificial inoculation, developed at IRRI (Ling, 1967, 1969), permits the inoculation of about a thousand seedlings a day in the greenhouse. It was further modified in 1972 to double the screening capacity. The method-with or without modifications-has been used for artificial screening of the germ plasm collections in India (Raychaudhuri and John, 1970), Indonesia (Rivera et al., 1968), Malaysia (Ou et al., 1965; Singh, 1969), Thailand (Wathanakul and Weerapat, 1969). Some varieties identified as resistant on the basis of greenhouse screening in various countries are listed in Table VI. In general, there is good agreement between the results from the field and greenhouse methods of screening. Both methods showed several varieties in each

TABLE VI Varieties Identified to Be Resistant to Tungro in Different Countries on the Basis of Field Tests and Artificial Screening in the Greenhouse Philippines Greenhouse Adday selection Badshahbog T 412 Basmati 370 Bengawan C 18 DV 29 Fadjar FB 24 Gam Pai-30-12-15 HR 21 Habiganj D.W.8 Latisail M. Sungsong Mas Pankhari 203 Peta Ram Tulasi Sigadis Tilakkachary Tjahaja Tjeremas IR 28 IR 29 IR 30 IR 34

India Field

Nagkayat W1263 Basmati 6 129 Ptb 10 Ptb 18 Hashikalmi Kamod 253 Kataribhog Gam Pai 15 HR 21 Habiganj D.W.8 Latisail M. Sungsong BJ 1 Pankhari 203 Peta RamTulasi Sigadis ADT 25 Ambemohar 105 Ambemohar 102 IR 28 IR 29 IR 30 IR 34

Greenhouse Ambemohar 159 Bhadas 1303 Intan IR20 Kamod Kataribhog Latisail NC 1626 NSJ 198 Pankhari 203 Sigadis Tilakkachary

Indonesia

Thailand

Field

Greenhouse

Field

Greenhouse

Ambemohar 159 Bhadas 1303 Intan IR20 Kamod Kataribhog Latisail ARC 13820 ARC 13901 Pankhari 203 Sigadis Tilakkachary Ptb 18 ARC 13677 ARC 13959 ARC 10342 IR 30 IR 32

Dara Pankhari 203 Peta Syntha

Cam Pai 15 Pankhari 203 Peta Latisail Habiganj D.W.8 Ptb 18 Ptb 21 Hashikalmi M. Sungsong ARC 7140 H8 IR 28 IR 29 IR 30 IR 32 IR 34

Bengawan H4 Latisail Pankhari 203 Peta Sigadis Tjeremas

Field Bengawan Muey Nawng 6 2 M Latisail Pankhari 203 Peta Sigadis Tjeremas

29 2

GURDEV S. KHUSH

country as resistant. Some discrepancies exist, however. Variety Ptb 18 is highly resistant in the field method of screening in the Philippines, but moderately susceptible in the greenhouse test. It has the Glhl gene for resistance to green leafhopper, and thus has high resistance to that vector. It would appear that Ptb 18 is not resistant to the virus but escapes infection under field conditions because of its high level of resistance to the vector. Some varieties-Pankhari 203, Latisail, and its progenies (Peta, Bengawan, and 1ntan)-are resistant in all countries. Susceptible varieties such as TN1, IR8, and IR22 have shown susceptibility in the same countries. Some varieties, such as Cam Pai 15, have shown differential reaction in some countries. Gam Pai 15 and its progenies IR28 and IR34 are highly resistant in the Philippines and Indonesia, but only moderately resistant in Thailand and susceptible in India. Gam Pai 15, IR28, and IR34 are also resistant to the green leafhopper. The observed differences in tungro reaction of the varieties may be due to the different biotypes of the vector in those countries. The available information points to the gaps in our knowledge about the role of vector resistance in virus control, strain variations of a virus from one country to another, and variations in the biotypes of the vector. c. Inheritance of Resistance. No thorough analysis of the mode of inheritance of tungro resistance has yet been carried out. Preliminary reports indicate that two genes may convey resistance in some varieties. A study of the cross Pankhari 203/Taichung Native 1 at IRRI indicated that resistance in Pankhari 203 is governed by two complementary dominant genes (IRRI, 1967a). According to Shastry ef al. (1972b) resistance in Latisail is under duplicate gene control. d. Breeding for Resistance. The earliest and classic work on breeding for tungro resistance was done in Indonesia in the early 1930s. Mentek disease-now known to be identical to tungro-has caused serious crop losses in that country since 1859. Even though its exact nature was not understood at that time, several varieties were found resistant to it when grown in rnentek-infected areas. At Bogor in 1934, Van der Meulen crossed the resistant variety Latisail from India with the susceptible variety Tjina from China. Seeds from the F4 bulk of that cross were divided into four parts and grown at four experiment stations located in the regions where mentek was causing serious crop losses. Three stations were in West Java, the fourth was in East Java. Individual plant selections were made at each station on the basis of resistance to mentek. Single plant progeny lines were further evaluated for mentek resistance, yielding ability, and agronomic traits at the stations and in farmers’ fields. From the trials, several advanced generation breeding lines with mentek resistance and good agronomic traits were identified and released as varieties. Selected were Bengawan (at Ngale in East Java), Peta, Mas, and Intan (at Singamerta), Fadjar (in West Java), Pelopor and Salak (Bogor), and Tjahaja (at Tjitajam, near Bogor).

DISEASE AND INSECT RESISTANCE IN RICE

293

Intan and Mas were released for commercial production in 1940; Tjahaja, Fadjar, Pelopor, Bengawan, and Peta, in 1941; and Salak, in 1942. Within a short time the varieties were distributed all over the country, where they gradually replaced the old, susceptible varieties, particularly where mentek was known to be serious (Van der Meulen, 1951). Varieties Peta, Intan, Bengawan, and Mas became especially popular in Indonesia and in the Philippines. Greenhouse tests in Indonesia, the Philippines, Thailand, Bangladesh, and India showed the four varieties to be resistant to tungro. The breeding program for tungro resistance at IRRI was started in 1966-1967. Several resistant varieties-Peta, Intan, Sigadis, TKh46, HR2 1, Malagkit Sungsong, Gam Pai, Ptb 18, Pankhari 203, and BJ1-were donor parents. Improved plant-type breeding lines with tungro resistance were identified from the crosses of most of those parents (Table VII). Seven IRRI named varieties are moderately to highly resistant to tungro. IR20, IR26, and IR30 inherit their moderate TABLE VII Some Improved Plant-Type, Tungro-Resistant Varieties and Selections Developed a t IRRI from Various Donor Parents Donor parent

Variety/Selection

Cross

TKM6

IR20 IR26 IR30

Peta3/TN1//TKM6 IR24/TKM6 IR24/TKM6//IR2Q4/Oryza nivara

Gam Pai 15

IR833-6-2 IR28

Peta3/TN1//Gam Pai 15 / Peta3/TN1//Gam Pai 15/4/IR8/Tad~kan//TKM6~ T N l ///IR244 10.nivarq Peta3/TN1//Gam Pai 15/4/IR8/Tadukan//TKM6' / TN1///IR244 10. nivara Peta3/TN1//Gam Pai 15/4/IR8/Tadukan//TKM6' / TNl///IR24" 10. nivara

IR29 IR34

Ptb 18

IR1702-7 IR2070-423-2 IR32 IR36

IR24/Ptb18 IR20210.nivarallCR94-13 IR20210. nivarallCR94-13 I R 8 / T a d ~ k a n / / T K M 6/TNl ~ ///IR244 10. nivara/4/ CR94-13

HR21

IR1364-37-3 IR2034-289-1

Peta3 ITNlIIHR21 IR24//Mudgo/IR8///Peta3 /TNl//HR21/4/IR244/ 0. nivara

Pankhari 203

IR825-11-2

IR8/Pankhari 203//Peta6 /TNl

Sigadis

IR127-80-1

CP23 l/SL017//Sigadis

294

GURDEV S. KHUSH

resistance from TKM6. Cam Pai 15 is the donor parent of highly resistant IR28, IR29, and IR34. The IRRI breeding program for resistance was expanded when we started screening the breeding materials under field conditions. The first extensive field test was conducted at four locations in the Philippines during the tungro epidemic in the 1971 wet season (IRRI, 1972). We screened a large number of breeding lines during that year. Since then we have screened all breeding materials under field conditions every year. We also screened several hundred advanced breedmg lines in Indonesia at Agricultural Sub-station Lanrang, South Sulawesi, in cooperation with Indonesian scientists in 1972-1973. As a result of the extensive field tests, IR26, IR28, IR29, IR30, IR32, and IR34 were identified as resistant. Greenhouse tests confirmed their resistance. Tungro resistance is one major breeding objective in Indonesia, Malaysia, the Philippines, Thailand, Bangladesh, and India. Variety C4-63, developed at the College of Agriculture, University of the Philippines, is moderately resistant to tungro. RD5 (Thailand) and BR4 (Bangladesh) are moderately resistant. Out of more than 50 high yielding varieties released in India, Triveni is highly resistant; Vijaya, Ratna, and Pusa 2-21 are moderately resistant. Several new, resistant, improved plant-type lines-CR110-173, CR139-1044-A25, CR138-999-A12, CR44-1048-Al2, and RP423-A24-have been developed in India; some are likely to become named varieties.

2. Grassy Stunt Disease Grassy stunt, which some researchers believe is caused by mycoplasma, was considered a minor rice disease until recently. Agati et al. (1941) in the Philippines described diseased plant symptoms which look like those of grassy stunt. However, the first diagnostic reports on grassy stunt are those of Rivera et al. (1966) and Bergonia et al. (1966) from the Philippines. The latter report called it rice rosette. On the basis of disease symptoms, vector species, and virus vector interactions, the diseases described in the two publications are the same. The disease was first observed at the IRRI farm in 1963 and was artificially transmitted by Nilaparvata lugens in 1964 (Rivera et al., 1966). Grassy stunt is now known to occur in Thailand (Wathanakul et al., 1968), Sri Lanka (Abeygunawardena et al., 1970), India (Raychaudhuri et al., 1967; Gopalakrishnan et al., 1973; Anjaneyulu, 1974), Malaysia (Ou and Rivera, 1969), and Indonesia (Tantera et aL, 1973). Some disease symptoms observed by Toan (1969) in Vietnam are similar to those of grassy stunt. The present author observed plants with grassy stunt in the delta provinces of Vietnam during several trips. Several varieties and breeding lines with resistance to grassy stunt in the Philippines were found to be resistant to the disease in Vietnam. Thus, there is n o doubt about the existence of the disease in Vietnam. The disease probably occurs in Cambodia, Laos, and Burma as well (Fig. 2).

DISEASE AND INSECT RESISTANCE IN RICE

295

a. Strain Variation. Differences in grassy stunt strains from one country to another have not been reported. So far, the reactions of several varieties to grassy stunt in the Philippines, Vietnam, India, and Indonesia are identical. IR8 and Taichung Native 1 are susceptible, but breeding lines derived from crosses of Otyza niuara are resistant. b. VarietalResistance. A large number of varieties were screened at IRRI for their reaction t o grassy stunt under field conditions, and in the greenhouse with an artificial mass screening technique developed at IRRI (Ling et al., 1970). Several varieties-BP176, Emata, H8, H105, H W 5 , Khao Dawk Mali 4-2-105, Khao Selti, Leuang Hawn, Puang Nahk 16, and TKM6-had fieldtolerance to the disease (Khush, 1970). Out of 6723 accessions of cultivated rice and several wild species of Oryza that were evaluated with the mass screening technique, only one accession of O y z a nivara was found highly resistant (Ling et al., 1970). To date, it is the only known source of resistance to grassy stunt. c. Inheritance of Resistance. A single dominant gene Gs confers resistance to grassy stunt in Oryza nivara (Khush and Ling, 1974). Gs segregates independently of Bphl, the dominant gene for resistance to brown plant hopper (vector of grassy stunt). d. Breeding fur Resistance. Oryza nivara, which has very poor plant type, grows as a weed in Central India. It has weak stems, a spreading growth habit, shattering panicles, long awns, and red pericarp. It is a poor yielder. In 1969 we crossed 0. nivara with IR8, IR20, and IR24. The F1 plants from the crosses were backcrossed four times using IR8, IR20, and IR24 as recurrent parents, respectively. For each successive backcross, F plants with morphological resemblance to the recurrent parents were selected. By the end of 1970, we had grassy stunt-resistant breeding lines resembling IR8, IR20, and IR24. However, lines lacked some other desirable traits, such as resistance to brown plant hopper and tungro. Therefore, they were used as parents in numerous crosses in 1971, 1972, and 1973. More than 80% of the crosses made in 1972 had at least one resistant parent. Segregating populations from these crosses were evaluated under field conditions at the IRRI farm where disease pressure was very severe in 1971-1974. The feasibility of rigorous field screening at IRRI enabled us to select numerous resistant lines that had multiple resistance to other diseases and insects and desired agronomic traits and grain quality. The first grassy stunt-resistant varieties IR28, IR29, and IR30 were released by IRRI in 1974, and IR32 and IR34 in 1975. IR2071-625, another resistant selection, was released as IR36 by the Philippine Government in 1975. The same selection was released to the farmers in Kerala state (India) in 1976. To combat the threat of grassy stunt, the Government of Indonesia approved the cultivation of IR28, IR29, and IR30 in 1975 under the names PB28, PB29, and PB30. IR34 is undergoing extensive testing in that country. Some promising, resistant lines developed at IRRI are listed in Table VIII. The lines have been sent to scientists in other countries where they are used in crossing programs as sources of grassy stunt resistance.

296

GURDEV S. KHUSH

TABLE VIII Some Improved Plant-Type Varieties and Breeding Lines with Grassy Stunt Resistance Developed at IRRI from Crosses Involving O v z a nivara Variety/Selection IR28 IR29 IR30 IR32 IR34 IR1721-11-6-8 IR1737-19-7-8 IR1825-31-3 IR1917-3-19-3 IR2031-238-5-2 IR2034-289-1-1 IR2035-290-2-3 IR2038-158-2-3 IR2042-101-2-2 IR2070-423-2-5 IR2076-67-3-5 IR2153-26-3-5 IR2564-103-2-1

Cross

Peta3/TN1//Gam Pai 15/4/IR8/Tad~kan//TKM6~ /TN1///1R244/ Otyza nivara Peta3/TNl//Gam Pai 15/4/IR8/Tad~kan//TKM6~ /TNl///IR24" / 0. nivara 1R24/TKM6//IR2O410.nivara IR202lO.nivarallCR94-13 Peta3/TNl//Gam Pai 15/4/IR8/Tad~kan//TKM6~/TNl///IR24~ / 0. nivara IR243 10. nivara IR24410. nivara IR 2 2 //I R 24 10. nivara IR20310. nivara IR24310.nivara/~lPeta4/TNl //Tetep/4/Leuang TawnglIR8lfW1263 1R24//Mudgo/IR8///Peta3ITNlIIHR21 /4/IR244 10.nivara Peta" /TN1//Tetep///Peta3/TNl//HR21/4/Mudg0/lR8/11IR24~/ 0. nivara IR24//Mudg0/1RS///IR24~10. nivara IR8/Tadukan//TKM6' /TN 1///IR24" 10. nivara IR2O210. nivarallCR94-13 IR24310. nivara///Peta' /TNl//HR21 IR24/TKM6//IR204 10.nivara Sigadisl /TNl//IR24///IR243 10.nivara

Several hundred resistant lines from IRRI were evaluated in Vietnam in the cooperative IRRf/Vietnam rice project, and some were being considered for varietal release at the time the government of that country changed in April 1975 (D. G. Kanter, personal communication). Besides the program at IRRI, active breeding programs on grassy stunt now exist in India and Indonesia. 3. Stripe Disease Stripe disease has been known in Japan since the early 1890s (Shinkai, 1962). It is widely distributed except in Hokkaido and the northern parts of Tohoku (Iida, 1969). It is widespread in South Korea (Lee, 1969) and may also occur in North Korea. It has been reported in mainland China (Chen, 1964) (Fig. 2). The disease is transmitted by the small brown plant hopper Laodelphax striatellus. a. Strain Variation. There are no reports on strain variation in the virus. Resistant varieties in Japan are aIso resistant in Korea; those susceptible in Korea are also susceptible in Japan.

DISEASE AND INSECT RESISTANCE IN RICE

297

b. Varietal Resistance. Resistant varieties have been identified in field trials where the test materials are planted early in the season (Suzuki et al., 1960). Through the seedling inoculation method devised by Sakurai et al. (1963), large numbers of native and introduced varieties were screened for resistance (Sakurai and Ezuka, 1964; Yamaguchi et al., 1965; Washio et a]., 1968a; Sonku and Sakurai, 1967). All Japanese lowland varieties as well as japonica varieties from Taiwan, Korea, China, Russia, and the United States were susceptible. However, the great majority of indica varieties from China, Southeast Asia, India, Pakistan, and Sri Lanka, and the bulu varieties from Indonesia were resistant to the virus. Included in the resistant group are important indica varieties such as Latisail, Intan, Peta, Tjahaja, Tadukan, PtblO, Danahara, Hatadavi, Tetep, and Charnak. In Korea the resistant varieties Arkrose, Gulfrose, Li Chan Chi1 I1 Chal, Nong Lim N01, Shin N02, and Zenith were identified from 410 varieties tested by the artificiaI inoculation method (Lee, 1969). Recently more local varieties, diseaseand insect-resistant indica varieties, and breeding lines from IRRI have been screened. U. R. Norinmochi 1, U. R. Norin 6, U. R. Jeunjageumna, and almost all breeding lines from IRRI are resistant (G. S. Chung, personal communication). c. Inheritance of Resistance. The inheritance of resistance was investigated by studying the F1, backcross, and F2 and F3 populations of crosses between susceptible Japanese lowland varieties and resistant Japanese upland varieties. The artificial inoculation technique was used in screening the hybrid populations. The results revealed the presence of two complementary dominant genes for resistance, Srl and St2, in Japanese upland varieties. Stl showed linkage with wx for the glutinous endosperm and with gene Se for photoperiod sensitivity. The gene Stl belongs to linkage group I. St2 was assigned to linkage group V (Washio et al., 1968a,c; Toriyama, 1969). A single incompletely dominant gene was found to confer resistance to indica as well as to bulu varieties from Indonesia. It is allelic to St2 but conveys a higher level of resistance than St2. It was therefore designated St2j. Stl shows complementary gene action, not only with St2 but also with St2' (Washio et al., 1968b,c; Toriyama, 1969). d. Breeding for Resistance. For developing stripe-resistant commercial varieties, the resistant upland variety Kanto 72 was crossed with the susceptible lowland variety Koshihikari. The F1 of the cross was topcrossed with another lowland variety, Kusabue. The F, plants from the three-way cross were artificially inoculated, and the resistant plants were again crossed with varieties Chuseishin-sengon and Kibiyoshi. After a thorough screening for several generations of the segregating populations from the crosses, four resistant varietiesChugoku 40, Chugoku 41, Chugoku 42, and Chugoku 49-were selected. The resistant varieties have the same yielding ability as the susceptible check variety (Toriyama, 1969, 1972).

298

GURDEV S. KHUSH

To transfer the incompletely dominant gene for resistance from indica varieties to japonica varieties, Toriyama et al. (1966) crossed Norin 8 with the Pakistani variety Modan, which was known to be resistant. The F1 was backcrossed five times to Norin 8 as recurrent parent. Of the 570 lines from the fifth backcross, Stl and Chugoku 31 were selected as stripe-resistant lines. Stl and Chugoku 31 were crossed with Sachi-hikari and Sachikaze, respectively. Variety Shimahashirazu was developed from the former cross, and Chugoku 46, Chugoku 5 1, and Chugoku 56 from the latter. These varieties have superior resistance to stripe and possess desired agronomic traits (Toriyarna, 1969, 1972). Variety Ton@ (IR667-98), developed from cooperative efforts between Korean scientists and IRRI, is highly resistant to stripe in Korea. It is planted in 30% of the rice area in that country. Because it was used extensively in the crossing programs, many breeding lines developed in Korea are also resistant to stripes. The promising selection Milyang 15, developed at Yungnam Crops Experiment Station, from a cross of Norin 6 and Chugoku 46, is highly resistant. 4. Dwarf Disease

Rice dwarf is the most widely known plant virus disease in the world. It was the first plant virus disease that was found to be carried by an insect, and it provided the first evidence for the multiplication of a plant virus in an insect. It is transmitted by the green rice leafhopper (Nephotettix cincficeps)-the major vector of the disease-and the zigzag leafhopper (Recilia dorsalis). The disease occurs in most parts of Japan, except Hokkaido (Iida, 1969), in Korea (Park, 1966), and probably in mainland China (Siang, 1952; Ou, 1972) (Fig. 2). No variation in the virus strain has been reported. Several varieties with high level of resistance have been identified through field tests and artificial inoculations. All Japanese lowland and upland varieties were found susceptible. Field tests showed varieties Hyakunichi-to-, Pe Bi Hun, Tetep, Loktjan, Kaladumai, and Dharial to be resistant (Sakurai, 1969). Artificial inoculation tests (Kimura er al., 1969; Ishii et al., 1969) showed the following varieties as resistant: Bluebonnet, C203-1, Chiem Chank, Dharial, Depi, Intan, Kaeu N525, Kaladumai, Karalath, Loktjan, Peta, Tadukan, and Tetep. According to Ishii et al. (1969), some resistant varieties are also resistant to the vector. Tetep, Tadukan, and TKM6 were found highly resistant in Korea (G. S. Chung, personal communication). The inheritance of resistance to dwarf disease has not been studied. Breeding for resistance is under way in Japan (Toriyama, 1972). Major emphasis is now placed on breeding for resistance at Yungnam Crops Experiment Station, Milyang, Korea. Several improved plant-type lines from IRRI, with Tadukan, Tetep, and TKM6 in their parentage, are resistant to dwarf disease in Korea. They are being used as sources of resistance in the crossing program.

DISEASE AND INSECT RESISTANCE IN RICE

299

5. Hoja blanca Hoja blanca is the only virus disease of rice reported from the Western Hemisphere. It occurs in all rice-growing countries of North, South, and Central America and the island nations of Cuba and Puerto Rico. It ranks next only to blast in importance in Latin America (Ou, 1972). The virus is transmitted by two species of plant hoppers: Sogatodes olyzicola (Muir) and Sogarodes cubanus (Crawf.). Although the disease was known and described in Colombia as early as 1935 (Galvez, 1969), it first came into prominence during the disease outbreak of 1956 in Venezuela (Atkins and Adair, 1957). a. Strain Variation. A uniform hoja blanca nursery with about 100 varieties and advanced generation breeding lines was grown at one or more locations in eight countries for several years. The varietal reactions at different locations showed no evidence for the existence of virus strains (Beachell et al., 1959; Lamey et al., 1963). To further explore the possibility of strain variation, a hoja blanca virus strain nursery of 18 resistant and susceptible varieties was tested under field conditions for 3 years in Colombia, Guatemala, and El Salvador. The tests indicated that the single strain present in Latin America before 1961 was the same strain present or dominant in Columbia in 1961 to 1963 (Lamey et al., 1963,1964b). b. VarietalResistance. To meet the threat of hoja blanca, a crash program of screening varietal collections for resistance was launched by the U.S. Department of Agriculture in 1957. A total of 2200 varieties and breeding lines of rice were field tested under epiphytotic conditions in Venezuela and Cuba, and an additional 1725 lines were tested in Cuba (Atkins and Adair, 1957). Most United States commercial rice varieties, United States rice germ plasm collection, and most F A 0 genetic stocks of rice were included in the tests. Most commercial United States varieties and indica varieties from Southeast Asia and Latin America were found susceptible. Many resistant varieties were identified. They included japonica varieties from China, Japan, Korea, Taiwan, Italy, Spain, and Portugal. A few indica varieties from India and Indonesia were resistant. Some United States medium- and short-grain varieties derived from japonica types were also resistant. Some resistant varieties identified in those tests (Atkins er al., I961a,b) are listed in Table IX. A technique for mass screening by artificial inoculation in the greenhouse was developed by Lamey et al. (1964a). It facilitated the greenhouse testing of breeding materials during the off season. The results in the greenhouse screening tests agreed with those in field tests. c. inheritance of Resistance. The inheritance of resistance was investigated by Beachell and Jennings (1961). From the data on the reaction of F1, backcross, and F3 populations of resistant and susceptible parents, it appears that a single dominant gene conditions resistance to the virus.

300

GURDEV S. KHUSH TABLE IX Some Hoja-Blanca-Resistant Varieties Identified from Field Tests Variety

C.I. or P.I. number‘

Country of origin

Pandhori No. 4 Mas Peta Bengawan co 10 N. 10-B S 67 Salak Tjahja Colusa Early prolific Gulfrose Lacrosse Missouri R 500 Tainau iku No. 487 Sadri

C.I. 6001 P.I. 181975 P.I. 181976 P.I. 193153 P.I. 193177 P.I. 197610 P.I. 233894 P.I. 220750 P.I. 220754 C.I. 1600 C.I. 5883 C.I. 9416 C.I. 8985 C.I. 9155 P.I. 215936 P.I. 184675

India Indonesia Indonesia Indonesia India India India Indonesia Indonesia United States United States United States United States United States Taiwan Iran

‘C.I. numbers refer to Cereal Investigations accession number and P.I. numbers refer to Plant Introduction number, both of the U.S. Department of Agriculture.

d. Breeding for Resistance. Most early work on breeding for resistance was done in the United States. Many crosses were made using Lacrosse, Gulfrose, Arkrose, PI21 5936 (Tainan-iku N0487), and Colusa as resistant parents. Sadri and Pandhori NO4 were used in a limited number of crosses. Segregating populations from the crosses were evaluated for resistance in Latin American countries, mainly at Palmira, Colombia. Some seeds from the pedigree selections were planted in the United States, and some were planted at Palmira to determine disease reaction. Further selections were made in the United States from the promising rows that showed resistant reactions at Palmira. The process was repeated until true breeding lines were obtained (Lamey, 1969). Several resistant varieties such as Northrose, Nova, and Nova 66 were developed (Johnston et al., 1965, 1966). Several highly resistant varieties were also bred in Colombia; ICAlO (Rosero, 1972) is the most resistant. Several breeding lines from IRRI, with Mudgo in their parentage, are highly resistant to hoja blanca (P. R. Jennings, personal communication). A large number of improved plant-type breeding lines were evaluated for hoja blanca resistance under field conditions in Cuba by J. Perez Ponce and his colleagues (personal communication). Varieties IR5, IR20, and IR24 were found highly resistant. Several lines from IRRI crosses-IR827, IR837, IR1108,

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301

IRllO9, IRl110, and IR1112-were also highly resistant. IR262-43-8-11, a breeding line from the cross Peta3 /TN1, is the common parent of all the crosses and is perhaps the resistant parent. Several resistant lines have excellent grain quality, the desired growth duration, and high yielding ability under Cuban conditions. A few lines are being considered for release as commercial varieties. More than 90% of the rice area in Cuba is planted to IR8 and IR160-27, both of which are susceptible to hoja blanca. The newer lines will be good replacements for the susceptible varieties.

I l l . Insect Resistance

More than 100 species of insects infest and feed on the rice crop. About 20 of them are of major economic importance. Considerable yield losses are caused by different species of stem borers, plant hoppers, leafhoppers, gall midge, and several other insects every year in most of the rice-growing areas of the world. Until a few years ago, chemicals were the only known means of insect control in rice. However, clear-cut cases of host resistance to several important insect species harmful to rice have now been identified. The high levels of resistance have been speedily incorporated into varieties with improved plant type. However, the sources of resistance to several important insect species are still not available and more research in this area is essential.

A. STEM BORERS

Stem borers are generally considered to be the most serious rice pests. They occur regularly and attack the crop at all stages of growth. They account for a large share of the crop losses caused by insects. Of the 20 species that attack the rice plant, four are of major importance in Asia. They are the striped borer, Chilo suppressah, the yellow borer Tryporyza incertulas, the white borer Tryporyza innotata, and the pink borer Sesamia inferens (Pathak, 1972). Research work on host resistance has been done mainIy for the striped borer and the yellow borer, and is reviewed here. Comprehensive reviews dealing with all aspects of stem borer research are those by Pathak (1968, 1969).

1. Striped Borer The striped borer is perhaps the most widely studied stem borer species. It is distributed in all countries of East Asia, Southeast Asia, South Asia, Egypt, and Spain. It is the dominant species in Japan, parts of the Philippines, Vietnam, several areas of the Indian subcontinent, Egypt, and Spain.

302

GURDEV S. KHUSH

a. Biotype Variation. Biotype variation in the insect through differential reaction in varieties has not been reported, and no critical work has been done on the subject. Kiritani and Iwano (1967) have reported the existence of three ecotypes of the insect in Japan. The ecotypes would perhaps show similar reactions on a set of rice varieties. b. Varietal Resistance. A large number of rice varieties have been screened for resistance to stem borers under conditions of natural infestation in India, Japan, and at IRRI. The work done in India was reviewed by Israel (1967); in Japan, by Munakata and Okamoto (1967); and at IRRI, by Pathak et al. (1971). The differences in resistance among varieties are quantitative in nature. Very high levels of resistance have not been found. There is continuous variation for this trait, among rice varieties-from highly susceptible to moderately resistant. Even those varieties classified as resistant suffer some damage under high insect populations. Matsuo (1952b), who screened 138 varieties of Japanese and foreign origin, found that foreign varieties were more susceptible than the Japanese varieties. That study and works by Fukaya (1947), Seko and Kato (1950), Tsutsui (1951), Ishikura and Watanabe (1955), and Okamoto and Abe (1958) identified several resistant Japanese varieties (Table X). Screening work for resistance to stem borers has been done in India since the early 1950s. Germ plasm collections have been screened at CRRI and several state experiment stations. Varieties C025, C019, ADTl, ASD8, ADT25, and ASD2 were reported resistant in Tamil Nadu; MTUl5, China 47, and Chautukalu were found resistant in Hyderabad. Bhamanik, Latisail, Raghusail, Nagra, Badshahbhog, and Patnai 23 had low stem borer incidence in Bengal. As a result of investigations at CRRI in 1951-1955, 36 varieties with low borer infestation were identified. Those that consistently showed low infestation (Israel, 1967) are listed in Table X. TKM6, MTUl5, and SLO12 were noted as being outstanding in resistance. In tests at Cuttack (CRRI, 1959) the varieties T1569 and AC546-143 showed high susceptibility at the vegetative or deadheart stage, but were resistant at the flowering or whitehead stage. Pathak et al. (1971) reported that some varieties are resistant at both stages, some are resistant at deadheart stage but not at flowering stage, a few others are susceptible at the deadheart stage but resistant at the whitehead stage. Several hundred varieties collected from northeast India were screened by AICRIP at Warangal in fields heavy with stem borer populations; 17 varieties were classified as resistant, but the stem borer species at the test site was not mentioned (Seetharaman et al., 1972). The largest amount of germ plasm (over 10,000 rice varieties and selections) was screened at JRRI between May 1962 and April 1965, under field conditions (Pathak et al., 1971). From the general trials, 1351 varieties were selected for further evaluation. They were replanted in one test in replicated trials. Varieties

303

DISEASE AND INSECT RESISTANCE IN RICE TABLE X Some Varieties Found to Be Resistant to Striped Borer in Different Countries Japan

India (CRRI)

Philippines (IRRI)

Kinmaze Kinki NO 25 Norin NO 23 Mihonishiki Nakate Ashahi Yubae Aichi Asahi Hatsushimo Benisengoku Kinki NO 24 Norin NO 6 Norin NO 8 Asahi Norin NO 22 Oboro Mock Y amadanishiki

TKM6 SLO 12 MTU 15 China 4 1 TKM 3 CO 13 BJ 1 China 51 TR3 D13 D14 AC 1971 ADT 1 8 SLO 6 Sajira MTU 20

Taitung 16 TKM6 Ti Ho Hung PI 160 Chaing an Tsao Pai Ku Patnai 6 Bir-co 884 co 21 HBJ Boro I1 C409 Su yai 20 Szu miao DNJ 97 DZ 41 DD 48 DV 88

‘The stem borer populations under field conditions in Japan and at IRRI are comprised primarily of the striped borer but at CRRI, India, the populations are composed of high proportions of striped borer and yellow borer as well as small numbersof other borer species.

selected from those trials were retested for differential reaction at deadheart and whitehead stages. The tests yielded about 60 varieties for further study. The selected varieties were tested under greenhouse conditions by infesting them with a uniform number of insects. Those that were found resistant in the field tests were usually resistant in the greenhouse tests. Some varieties found resistant on the basis of the extensive trials are listed in Table X. c. Inheritance of Resistance. Reports on the inheritance of resistance to stem borer are only fragmentary. Using borer infestation as a criterion of resistance, Koshiaty et al. (1957) showed that the field resistance of Giza 14 to stem borer was under polygenic control. The field resistance of TKM6 to stem borer as measured by the incidence of whitehead was reported to be simply inherited (AICRIP, 1968). One drawback of genetic studies under field conditions is that stem borer populations are generally composed of several species; resistance to one species may not mean resistance to the other species. Consequently, even a small admixture of species may nullify the genetic interpretations. The possibility of escapes and nonuniform population pressures are other sources of error. Athwal and Pathak (1972) studied the inheritance of resistance in the greenhouse. Each of the 133 Fz plants from the cross between Rexoro (susceptible) and TKM6 (resistant) were infested with 10 larvae of striped borer. Plant

304

GURDEV S. KHUSH

reaction was determined according to the survival rate and body weight of the larvae. The resistance was dominant in the F1 . Larval weight was independent of survival rate and was used as an index of resistance to stem borers. From the frequency distribution of mean body weight of surviving larvae on the F2 plants, it was concluded that that particular component of resistance to stem borers may be simply inherited. However, the authors felt that more detailed studies are needed to draw definite conclusions about the various components of inheritance. d. Breeding for Resistance. Several crosses with stem-borer-resistant varieties were made at IRRI in 1966-1969. Some parents proved to be poor combiners and did not yield promising progenies. However, excellent progenies were obtained from the crosses of TKM6. A large number of improved plant-type selections were selected from those crosses and evaluated for agronomic traits, grain quality, and resistance to diseases and insects, including stem borers. Selected true breeding lines were evaluated for stem borer resistance in the greenhouse. From those crosses several important commercial varieties with stem borer resistance have been developed at IRRI as well as in other countries (Table XI). The varieties are now planted on millions of hectares of rice land and have been used extensively as parents in the crossing programs at IRRI and in other countries. It is of common knowledge that most widely grown improved-plant-type varieties, such as IR5, IR8, IR20, IR22, IR26, Jaya, Vijaya, Chandina, Mehren, Palman 579, show much less stem borer damage under field conditions than do tall, traditional, tropical varieties. Whether all those varieties have more genetic resistance than the tall varieties or get less stem borer infestation because of short stature is not fully understood. Perhaps both factors are involved. It is known that the dwarf varieties, Taichung Native 1 and Dee-geo-woo-gen, originally used as sources of short stature, have some resistance and have perhaps contributed some genes for resistance. The gene action for stem borer resistance seems to be additive. From the crosses of two moderately resistant lines or varieties, it is possible to obtain progenies with levels of resistance higher than those of their parents. IR747-B2-6, a breeding line from the cross T K l ~ l /TNl, 6 ~ has a much higher level of resistance than either TKM6 or TN1. The line was crossed with another moderately resistant line, IR579-48-2. Several lines from the crossIR1561-228-3, 1131561-243-5, and IR1561-250-2-have even higher levels of resistance. To exploit the situation, we started a breeding project to combine genes for stem borer resistance from several sources. IRRI entomologists have developed a screenhouse cage technique that allows for speedy evaluation of fairly large numbers of varieties under controlled conditions (IRRI, 1972). Through that technique several moderately resistant, improved plant-type lines have been identified. The lines originated from different parents and presumably

DISEASE AND INSECT RESISTANCE IN RICE

305

TABLE XI Some Striped Borer-Resistant Varieties and Breeding Lines Developed from TKM6 Crosses Made at IRRI Selection

Parents

Variety name

Country where named

IR532-1-18

Peta’ /TNl//TKM6

IR532

Sri Lanka

IR5 3 2-1-176

Peta’ /TNl//TKM6

Chandina

Bangladesh

IR532E208

Peta3/TNl//TKM6

NG6637

Papua New Guinea

IR53 2E5 76

Peta3/TN1//TKM6

IR20

Philippines, Vietnam, India, Bangladesh

IR580-25-3

IR8/TKM6

IR747B2-6

TKM6 * /TN 1

GPLl “Jumbo”‘

British Solomon Islands Philippines

IR1514AE597

IR20/TKM6

IR1541-102-7

IR24ITKM6

IR26

Philippines, Vietnam, Indonesia

IR1561-228-3

IR8/Tadukan//TKM6’ /TNl

TN73-2 Sakha 2 GPLS “IR1561”‘

Vietnam Egypt British Solomon Islands Philippines

‘These selections were not officially approved but are widely grown under popular names in the Philippines.

have different genes for resistance. Through a process of recurrent selection, we are trying to combine those genes to develop lines with a level of resistance hitherto unknown in rice. TKM6 has been used in several other breeding programs as a source of resistance to stem borers. Several varieties such as Ratna have been developed in India from TKM6 crosses. Several lines from the cross of IR8 and TKM6 made at AICRIP-IET 2812 and IET 3093-have very good levels of resistance.

2. The Yellow Borer The yellow borer, Trypolyzu incertulus ranks with the striped borer in importance. It is distributed in East Asia, Southeast Asia, the Indian subcontinent, and Afghanistan. It is a dominant stem borer species in Indonesia, some parts of the Philippines, and vast areas of the Indian subcontinent.

306

GURDEV S. KHUSH

Not much information is available on varietal resistance to the pest. Varietal collections at three research centers are being evaluated for resistance to the yellow borer. One thousand varieties and breeding lines were evaluated in the screenhouse at IRRI in 1973-1974, and 1250 varieties and breeding lines were tested under field conditions in Maligaya Rice Research and Training Center in Central Luzon, Philippines, in the dry season of 1975. The yellow borer is the predominant species in the dry season in that part of the country. In the tests, 56 and 80 entries had low borer infestation. Among selected entries, which were reevaluated in the greenhouse, several varieties and improved plant-type breeding lines showed moderate levels of resistance. Most outstanding of them are IR1820-52-2, W1253, W1263, IR1917-3-19, IR1721-11-6-8, Kipusa, TKM6, and IR1539-823-1-4 (I. Manwan and M. D. Pathak, personal communication). Varieties that showed good levels of resistance under field conditions at CRRI include W1263, MNPl19, CR43-76, and Ptb 18 (P. S. Prakasarao, personal communication). In Indonesia, several hundred varieties and breeding lines have been screened under field conditions at Pusakanegara where severe yellow borer infestation occurs regularly. Varieties ARC10257, ARC10598, ARC10692, W1251, W1257, W1263, and C022 from India; Leri, Dendek kolon, and Pelopor from Indonesia; and breeding lines IR1330-90-2 and IR1330-51-1 from IRRI consistently showed low borer incidence in replicated screening trials ( C . Van Vreden, personal communication). Fernando (1 967) reported Kalu Heenati, H4, and H5 to be resistant in Sri Lanka. No work on breeding for resistance to yellow borer has been done to date, since sources of resistance are not available. However, some resistant entries identified recently have been included in the hybridization programs.

1. Brown Plant Hopper Plant hoppers were considered minor pests of rice until a few years ago. At the symposium on “The Major Insect Pests of the Rice Plant,” held at IRRI in September 1964 (IRRI, 1967b) only one of 36 papers was devoted to plant hoppers (Nasu, 1967). At present, plant hoppers constitute a major threat to rice production in most main rice-growing areas of the world. Plant hoppers infest and multiply in the basal part of the plant. H~ghtemperatures and high humidity are ideal for their multiplication. Conditions that produce excellent hosts include : high tillering, improved plant type varieties with dense vegetation (especially when planted at closer spacing), high level of fertility, and good weed and water control. The wide-scale adoption of high yielding varieties and intensive management practices in recent years have contributed to the sudden outbreaks of plant hoppers in many countries of the world.

DISEASE AND INSECT RESISTANCE IN RICE

307

Light plant hopper infestations reduce tillering, plant height, number of productive tillers per plant, and general vigor of the crop, and increase the number of unfilled grains. Heavy infestations can destroy the crop completely by producing the condition known as “hopperburn.” Plant hoppers also act as vectors of important viruses and thus cause yield losses indirectly by spreading diseases. Increased emphasis is now put on host resistance to plant hopper in many rice improvement programs. Several plant hopper species are known to attack the rice crop. Four are of major importance and available information on resistance is reviewed here.

1. Brown Plant Hopper The brown plant hopper, Nilupurvatu lugens Stal., is the most destructive insect pest of rice today. It occurs in South, Southeast, and East Asia, and Micronesia (Fig. 3). Cases of hopperburn and serious yield losses caused by the insect have been reported for many countries in recent years.

FIG. 3. Distribution of brown plant hopper, white backed plant hopper, green leafhopper, rice green leafhopper, and small brown plant hopper in Asia. The gene center for resistance t o brown plant hopper is also shown.

308

GURDEV S . KHUSH

a. Biozype Variation. Until recently brown plant hopper populations were thought to belong to the same general biotype. Varieties found resistant at IRRI such as Mudgo, ASD?, and their progenies, were tested by local scientists in Korea, Japan, Taiwan, Vietnam, Thailand, Indonesia, British Solomon Islands, and Fiji and were found resistant to the native brown plant hopper populations. Initial reports from Sri Lanka also indicated that Mudgo was resistant in that country. The brown plant hopper outbreaks of 1973 and 1974 in India and Sri Lanka, respectively, generated much interest in varietal resistance to the insect in those countries and serious screening work was undertaken. It was found that the brown plant hopper populations in India and Sri Lanka belonged t o a different biotype, to which Mudgo and ASD7 are susceptible. Thus, two natural biotypes of the brown plant hopper are now known. Biotype 1 is present in all countries of East Asia, Southeast Asia, and Micronesia. The South Asian biotype is present in South India and Sri Lanka. Whether all parts of India and other countries of the Indian subcontinent-Nepal, Bangladesh, and Pakistan-have the same biotype is not known. The biotypic identity of the brown plant hopper populations of Burma and Malaysia is also unknown; the individuals perhaps belong to Biotype 1 . A third national biotype of brown plant hopper originated in the British Solomon Islands in 1974. Several resistant breeding lines were introduced and grown in that country. Although highly resistant, they became susceptible in the later part of 1974 because of the development of a new biotype. New biotypes have also been produced by rearing the insects on resistant varieties at IRRI (Athwal and Pathak, 1972) and in Taiwan (C. H. Cheng, personal communication). Two biotypes were collected by IRRI entomologists from fields planted to resistant varieties in the Philippines. One of them-called biotype 2-attacks the resistant variety Mudgo; biotype 3 attacks ASD7 (IRRI, 1975). The ease with which new biotypes are produced in the laboratory and the fast appearance of a biotype in nature under the influence of host resistance in the British Solomon Islands and the Philippines does not augur well for the continued success of host resistance in controlling the insect. A great challenge lies ahead for the entomologist and the plant breeder. b. Varietal Resistance. Differences in varietal resistance to brown plant hopper under field conditions were noted by Israel and Rao (1954). In a replicated trial of 14 varieties planted at CRRI in the monsoon season of 1953, a severe attach of brown plant hopper occurred. Varieties Ch. 2 , Ch. 41, Ch. 42, Ch. 43, Ch. 45, Ch. 47, Ch. 62, and Ch. 63 from China were severely attacked, but the local varieties Adt. 20, N. 136, Ptb 10, B76-1, Adt4, and Benibhog showed very little hopper population. Clear-cut differences in varietal resistance to brown plant hopper under greenhouse conditions were first demonstrated at IRRI in 1967 (IRRI, 1968; Pathak et a l , 1969). Since then several thousand varieties from the germ plasm bank at

DISEASE AND INSECT RESISTANCE IN RICE

309

IRRI have been evaluated, and more than 120 resistant varieties have been identified (Pathak, 1972; IRRI, 1975). The resistant varieties were screened for resistance to biotypes 2 and 3; more than 20 of them are resistant t o both (M. D. Pathak, personal communication). Some resistant varieties are listed in Table XII. More than 2000 varieties were screened for resistance in Taiwan. All local varieties were susceptible, but a number of introduced varieties were resistant (Chang and Chen, 1971; Chow and Cheng, 1971). The varietal reactions in Taiwan and at IRRI are in perfect agreement. Researchers at AICRIP have identified Ptb 33 and ARC6650 as resistant to the South Asian biotype (M. B. Kalode, personal communication). Ptb 33 is also resistant in Sri Lanka and to all the three Philippine biotypes; it is the only variety resistant to all the known biotypes of brown plant hopper. It is interesting that all resistant varieties that have been identified-with the exception of one or two-come from Sri Lanka and adjoining states of India (Tamil Nadu, Kerala, Karnatka, Andhra Pradesh, and Maharashtra). That gene center for brown plant hopper resistance is shown in Fig. 3. c. Inheritance of Resistance. The investigation of the inheritance of resistance to brown plant hopper by Athwal et al. (1971) showed that the resistance in varieties Mudgo, C022, and MTU15 was controlled by single dominant genes which appeared to be allelic to each other. The locus was designated Bphl. A single rececsive gene that conveyed resistance in ASD7 was designated bph2. Bphl and bph2 are closely linked and no recombination between them was observed. Chen and Chang (1971) also reported a single dominant gene for resistance in Mudgo. Athwal and Pathak (1972) reported that MGL2 and Ptb 18 possess Bphl and bph2, respectively. Two improved plant-type breeding lines-IR747B2-6 and IR1154-243-were found resistant under field conditions at IRRI (IRRI, 1970). None of the parents of the lines are resistant to brown plant hopper. Martinez and Khush (1974) showed that resistance in IR747B2-6 is governed by Bphl, and bph2 conditions resistance in IRll54-243. They found that TKM6 is susceptible to brown plant hopper, but when it is crosses with other susceptible varieties such as TN1, IR8, or IR24, a small number of the F2 progeny are resistant. It was hypothesized that TKM6 is homozygous for Bphl as well as for a dominant inhibitory gene I-Bphl, which inhibits Bphl. IR4-93, a dwarf line from the cross between H105 and De-geo-woo-gen, was found to have bph2 for resistance (Martinez and Khush, 1974). H105 is the resistant parent of that selection. To identify more loci for resistance to brown plant hopper, Lakshminarayana and Khush (1977) carried out genetic analysis of 28 resistant varieties. Nine had Bphl, and sixteen, bph2. Two new loci for resistance were discovered. A single dominant gene, which governs resistance in Rathu Heenati, segregates independently of Bphl and was designated Bph3. A single recessive gene, which conveys

310

GURDEV S. KHUSH

resistance in Babawee, segregates independently of bph2 and was designated bph4. Resistance in Ptb 21 is controlled by one dominant and one recessive gene. The allelic relationship of the two genes in Ptb 21 to the other four genes are not known, and further studies are in progress. Investigations on inheritance of resistance to brown plant hopper are also under way in Taiwan, at Chaiyi Agricultural Experiment Station. It was found that varieties MTU9, Sudurvi 306, and Murunga 137 possess single dominant genes for resistance; the allelic relationships of those genes to Bphl were not investigated. Resistance in IR9-60, Kaosen-yu 12, and H5 was governed by recessive genes which are allelic to bph2 (W.L. Chang and C. H. Cheng, personal communication). The resistant varieties that have been analyzed genetically to date, and the respective genes for resistance are in Table XII. d. Breeding for Resistance. Breeding for resistance to brown plant hopper was initiated as soon as the sources of resistance were identified. Mudgo and IR8 have poor grains. A cross made between them in 1967 produced progenies with poor grains. Therefore, an F3 line of the cross was hybridized with IR22 and IR24. Promising progenies such as IR1614-138-3 and IR1614-389-1, were selected from the cross with IR22, and IR1539-260 and IR1539-823-4 were selected from the cross with IR24. These lines have excellent grain quality, high yield potential, and resistance to brown plant hopper and green leafhopper; but they are susceptible to tungro, grassy stunt, and blast. Several F 3 and F4 selections of IR1539 were crossed with other promising breeding lines in 1970, and the F1s were crossed with other F, s in 1970 and 1971. From the double crosses IR2034, IR2035, and IR20.58, several very promising breeding lines with multiple resistance were selected and were evaluated as varietal possibilities (Table XIII). In 1969 a serious outbreak of brown plant hopper occurred at IRRI farm, and a yield trial of 55 early maturing entries had hopperburn. Two entriesIR747B2-6 and IR1154-243, with improved plant type and good grain qualityshowed resistance and did not suffer any damage. They were immediately crossed with other promising lines. The outstanding resistant lines, IR1561149-1, IR1561-228-3, IR1561-243-5, and IR1561-250-2, were selected from the cross of IR747B2-6 and IR579-48; IR1628-632-1, from the cross of IR24 and IRI 154-243 (Table XIII). The selections have high-yield potential, excellent grain quality, resistance t o several diseases and insects, but are susceptible to tungro and grassy stunt. In 1970, IR1561-149-1 was crossed with IR1737, a grassy stunt-resistant line from the fourth backcross of olyza nivara to IR24. The F1 was topcrossed in 1971 with IR833-6-2 (resistant to tungro and blast) line from the cross Peta3/TN1//Gam Pai 15. Progenies from that cross were thoroughly evaluated for resistance to all major diseases and insects, and for grain quality and agronomic traits. Two lines-IR2061-214-3-8 and

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TABLE XI1 Some Brown Plant-Hopper-Resistant Varieties and the Resistance Genes Possessed by Them Variety/Selection

Resistance gene

Reference

Mudgo MTU 15 co 22 ASD 7 MGL 2 PTB 1 8 H 105 IR1154-243 IR747B2-6 Anbaw C7 Tibiriwewa Balamawee co 10 Heenakkulama MTU 9 Sinnakayam SLO 12 Sudhubalawee Sudurvi 305 ASD 9 Dikwee 328 Hathiel Kosatawee Madayal Mahadikwee Malkora M. I. 329 Murungakayan 302 Ovarkaruppan Palasithari 601 PK-1 Seruvellai Sinna Karuppan Vellailangayan Rathuheenati Babawee Ptb 21 Samba H5 IR9-60 Kaosen-yu 12

Bph I Bph I Bph 1 bph 2 Bph I bph 2 bph 2 bph 2 Bph I bph 2 Bph I Bph 1 Bph I Bph I Bph I Bph I Bph 1 Bph I Bph I bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 bph 2 Bph 3 bph 4 7' bph 2 bph 2 bph 2 bph 2

Athwal et al. (1971) Athwal e f al. (1971) Athwal e f al. (1971) Athwal eta!. (1971) Athwal and Pathak (1972) Athwal and Pathak (1972) Martinez and Khush (1974) Martinez and Khush (1 974) Martinez and Khush (1974) Lakshminarayana and Khush (1977) Lakshminarayana and Khush ( I 977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1 977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush ( I 977) Lakshminarayana and Khush (1 977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Lakshminarayana and Khush (1977) Chang and Cheng (personal communication) Chang and Cheng (personal communication) Chang and Cheng (personal communication) Chang and Cheng (personal communication)

'This variety has two genes for resistance but their allelic relationships are not known.

312

GURDEV S.KHUSH TABLE XI11 Some Improved Plant-Type Brown Plant-Hopper-Resistant Varieties and Breeding Lines Developed at IRRI

Variety/Selec tion IR26 IR28 IR29 IR30 IR32 IR34 IR4-93 IR747B2-6 IR1154-243 IR1330-3-2 IR1539-823-4 IR1561-228-3 IR 1614-138-4 IR 1628-632-1 IRl702-74-3 IR2031-724-2 IR2034-289-1 IR2035-290-2 IR2038-15 8-2 IR2070-423-2 IR2071-625-1

Parents IR24/TKM6 Peta3/TNl//Gam Pai 15/4/IR8/Tadukan//TKM62/ TN1///IR244 /Oryza nivara Peta3 /TNl//Gam Pai 1 5 / 4 / I R 8 / T a d ~ k a n / / T K /6 ~ TN1///IR244 10.nivara IR24/TKM6//IR2O4/0. nivara IR20210.nivarallCR94-13 Peta3 /TNl//Gam Pai 15/4/IR8/Tadukan//TKM6' / TNl///IR244 10.nivara HlOS/Dgwg TKM6' /TN 1 IR8' /Zenith Leuang Tawng/IR8//W1263 IR24//Mudgo/IR8 IRI/Tadukan//TKM6 /TN 1 IR22//Mudgo/IR8 IR24flIR8' /Zenith IR24/Ptb 18 IR24 10.nivara///Peta4/TN 1//Tetep/4/Leuang Tawng/IRB//W1263 IR24//Mudgo/IR8///Peta3 /TN1//HR21/4/IR244 / 0. nivara Peta4 /TN1//Tetep///Peta3 /TNl//HR21/4/IR24// Mudgo/IR8///IR24 f0. nivara IR24//Mudg0/1RS///IR24~10.nivara IR20210.nivarallCR94-13 IRI/Tadukan//TKM6' /TNl///IR24410.nivara/4/ CR94-13

Resistance gene Bph I Bph 1 Bph 1 Bph I bph 2 Bph I bph 2 Bph I bph 2 Bph I Bph 1 Bph 1 Bph I bph 2 bph 2 Bph 1 Bph I Bph 1 Bph I bph 2 bph 2

IR2061-4644-from the cross were named IR28 and IR29, respectively, in 1974; a third, IR2061-213-2-17, was named IR34 in 1975. As mentioned earlier, when TKM6 is crossed with other susceptible varieties a small number of the segregating progeny are resistant to brown plant hopper. The cross of IR24 and TKM6 in 1969 produced two such lines-IR1541102-7-491 and IR1541-76-3. The former selection was named IR26 in 1973; it had multiple resistance to major diseases and insects, good grain quality, and high-yield potential. In 1971, IR1541-102-6, a close sib of IR26, was crossed with a plant of the fourth backcross of Oryza nivara to IR20, and produced the brown plant-hopper-resistant IR30. IR2070-747-6-3-2, a resistant line from the cross IR202/0. niuara//CR94-13 was named IR32 in 1975.

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As the various resistant breeding lines became available, they were included in the crossing program. Less than 10% of the F2 populations grown in 1969 were segregating for brown plant hopper resistance. The proportion steadily rose to more than 90% in1974. The 2% resistant entries in the replicated yield trials in 1969 rose to 98% in 1974. Special attention was paid to incorporating dominant as well as recessive genes for resistance into an improved plant-type background. About half of the entries in the replicated yield trials in 1974 had Bphl, and the other half, bph2. Of the IRRI named varieties IR32 has the recessive gene for resistance; the others have the dominant gene. IR36 and IR38, whch the Philippine Government named in 1976, have the recessive gene for resistance. We are already incorporating into varieties Bph3 and bph4, the resistance genes identified recently. Since Bph3 and bphl segregate independently of Bphl and bph2, we are also planning to develop germ plasm with two genes for resistance. Since 1969, IRRI has been supplying germ plasm of improved plant-type, brown plant-hopper-resistant lines to various countries where they have been used as parents in crossing programs. Some have been named as varieties: IRI 561-228-3-3 was named TN73-2 in 1973 in Vietnam. IR747B2-6 and IR1614-138-3 were named GPLl and GPL2, respectively, in the British Solomon Islands. The Government of Indonesia approved IR26, IR28, IR29, and IR30 in 1975 for cultivation in that country. Outside of the program at IRRI, the most active breeding program for brown plant hopper resistance is that in Taiwan. Scientists at Chaiyi Agricultural Experiment Station, who have systematically incorporated both genes for resistance into the indica as well as the japonica background, released the resistant variety Chianung Sen 11 in 1974. Korean scientists have been developing brown plant-hopper-resistant materials for the last 4 years, and several resistant lines are being evaluated. Resistance genes have also been incorporated into Japanese varieties in a backcrossing program (Kaneda, 1971). In the tropics, the breeding program in Tailand has incorporated resistance to brown plant hopper into many promising breeding lines; two brown plant-hopper-resistant varieties, RD4 and RD9, have been released. Numerous crosses have been made with resistant lines in Indonesia. Breeding programs for brown plant-hopper resistance were organized in India and Sri Lanka in 1974. Scientists in Bangladesh, Burma, and Malaysia need to intensify their efforts on brown plant-hopper resistance. Four countries are now growing varieties resistant to brown plant hopper. The largest area is in the Philippines, and most of it is planted to IR1.561-228-3-3, IR26, IR28, IR29, IR30, IR32, IR34, IR36, and IR38. In Vietnam, IR1561228-3, IR26, and IR30 are in great demand. More than half a million hectares were planted in 1976. In 1976, approximately a million hectares were planted to IR26, IR28, and IR30 in Indonesia. Estimates of area planted to Chianung Sen 11 in Taiwan are not available.

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2. White-BackedPlant Hopper The white-backed plant hopper Sogatella furcifera Horvath occurs in South Asia, Southeast Asia, East Asia, Micronesia, Brazil, and the Caribbean countries (for Asian distribution, see Fig. 3). It does great harm to the rice crop in the tropics and subtropics. Cases of hopperburn caused by the white-backed plant hopper have been reported in Malaysia (Yunis and Rothschild, 1967), Indonesia (S. Sama, personal communication), and India, where the pest is particularly serious in the states of Madhya Pradesh, Uttar Pradesh, and Punjab. The insect cohabits with the brown plant hopper in the basal part of the rice plant. On susceptible varieties, the brown plant hopper multiplies faster and maintains numerical superiority. On brown plant-hopper-resistant varieties, however, there is no competition, and the white-backed plant hopper multiplies rapidly. A very high population of this insect was observed on IR26 at IRRI farm in the 1974 wet season. No biotypic variation has been reported in this insect. Varietal resistance has been studied at IRRI, in Taiwan, and in Korea. The resistant varieties identified at IRRI (Pathak, 1972; IRRI, 1973) are listed in Table XIV. Varieties Murunga 137 and Panneti are resistant in Taiwan. A single dominant gene governs resistance of N22 (IRRI, 1977). Breeding for resistance is under way at IRRI. In a backcrossing program, we are using N22 as donor variety and IR28, IR30, IR32, and IR34 as recurrent parents. Breeding lines with multiple resistance to white-backed plant hopper and other diseases and insects would be available before the end of 1977. Scientists in Taiwan and Korea are also developing materials with resistance to this insect. 3. Rice Delphacid The rice delphacid Sogatodes olyzicola Muir is distributed in all the rice-growing areas of South and Central America, Southern United States, and the Caribbean island countries. It is the most serious rice pest in the Latin American countries. Besides causing direct feeding damage and occasional hopperburn, it vectors the hoja blanca virus, the cause of a most serious rice disease in the Western Hemisphere. During severe insect infestations in 1964 and 1965 in Colombia, damage from direct feeding reduced the rice yields to 600 to 800 kg/ha (Samper, 1968). Normal rice yields in the affected areas range from 3000 to 5000 kg/ha. In 1967 and 1968, scientists at Centro Internacional de Agricultura Tropical (CIAT) at Cali, Colombia, observed that small plots of IR8 were free of insects when the adjoining plots of other varieties, such as Bluebonnet and Dawn, were completely killed. Varietal reactions to rice delphacid were then studied under controlled conditions in the laboratory, and clear-cut differences for resistance

315

DISEASE AND INSECT RESISTANCE IN RICE TABLE XIV Some Varieties with Resistance to Plant Hoppers and Leafhoppers Sogatella furcifera

Sogatodes oryzicola

Nephotettix cincticeps

Laodelphax striatellus

Colombo N22 Pankhari 203 JBS 34 H5 SLO 1 2 Kaluheenati Sudhubalawee ARC5 15 2

IR8 IR24 Mudgo TKM6 Tip 32-1-5 CICA 4 CICA 6 Pankhari 203

Dharial Kaladumi Tadukan Tetep Mumnga 137 Pannet ti H 105 1R4-93

ASDI Vellailangalayan Murunga 13I Pannet t i Konanso Tadukan Hu-nau-tsao

were noted. Out of 534 varieties tested, about 20% were highly resistant, 40% were intermediate, and 40% were susceptible. None of the varieties from the Western Hemisphere are resistant. Of the 142 japonica varieties tested, 127 were susceptible, and 15 were intermediate. The resistant varieties were indicas from South and Southeast Asia (Jennings and Pineda, 1970). Some resistant varieties are listed in Table XIV. The inheritance of resistance has not been intensively studied. Jennings and Pineda (1970) noted that the trait is highly heritable. Resistance to rice delphacid is one of the most important breeding objectives of the rice improvement program at CIAT. Two varieties-CICA4 and CICA6, released jointly by CIAT and ICA (Instituto Colornbiano Agropecuari0)-are resistant to the insect.

4. The Small Brown Plant Hopper The small brown plant hopper Laodelphax striatellus Fallen is distributed in Taiwan, Japan, Korea, China, and the Palearctic regions (Fig. 3). It causes considerable direct damage to the rice crop in Taiwan, Japan, and Korea. It is the vector of rice stripe, the most serious virus disease of the East Asian countries. It also transmits the rice black-streaked dwarf virus. No biotype variation has been reported in the insect. Varieties ASD7 and Vellailangalayan are highly resistant to the insect in Korea (S. Y. Choi, personal communication). Murunga 137 and Pannetti are highly resistant in Taiwan (C. H. Cheng, personal communication). Konanso, Tadukan, and Hu-nan-tsao are resistant under field conditions in Japan (Okamoto and Inoue, 1967). Resistant varieties are listed in Table XIV. Inheritance of resistance has not been studied. Scientists in Korea and Taiwan are incorporating resistance to this insect into their breeding materials.

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GURDEV S . KHUSH

C . LEAFHOPPERS

Like plant hoppers, leafhoppers were considered only minor rice pests until a few years ago. They inhabit and feed on the upper parts of the plant. Considerable damage is caused by direct feeding, resulting in reduced number of tillers, plant height, general vigor of the crop, and the number of filled grains. Under heavy population pressure, the crop turns yellowish, simulating symptoms of nitrogen deficiency. Only a few cases of hopperburn caused by leafhoppers have been reported. Leafhoppers are vectors of major virus diseases of rice in Asia and cause considerable indirect damage by spreading the virus diseases. Considerable interest has been generated in host resistance to leafhoppers in recent years, especially following the outbreaks of virus diseases. Of several leafhopper species which attack the rice crop, two are of major importance at this time and information on host resistance to them is accumulating.

I . The Green Leaflopper The green leafhopper Nephorettix virescens is distributed in South and Southeast Asia as well as in Southern Japan and Taiwan (Fig. 3), but it does greater damage in the tropics. a. Biotype Variation. Biotype variation in the insect has not been reported. There are unconfirmed reports that Pelita I/1, w h c h is resistant to green leafhopper in the Philippines, is susceptible in Indonesia. When a set of differential varieties is screened for reaction to the local populations of the insect in several countries, differences in the biotypes are likely to be found. b. Varietal Resistance. Differences in varietal reaction to green leafhopper were first reported by Pathak et al. (1969). They found IR8 and Pankhari 203 resistant, and TNI susceptible. Twenty-four additional resistant varieties were reported by Cheng and Pathak (1972). A large number of varieties from the IRRI germ plasm bank have been evaluated, and more than 50 resistant varieties are known (IRRI, 1973). Some resistant varieties are listed in Table XV. Resistant varieties in India were identified by Shastry et al. (1971). Resistant germ plasm comes from several countries of South and Southeast Asia and most resistant varieties are indicas. c. Inheritance of Resistance. The inheritance of resistance was investigated by Athwal et al. (1971) in varieties Pankhari 203, ASD7, and IR8. The resistance in the three varieties was found to be under monogenic and dominant gene control. The dominant genes were designated Glhl (in Pankhari 203), Glh2 (in ASD7), and Glh3 (in IR8). The three genes segregate independently of each other. Ptb 18 has two genes for resistance, one of which may be allelic to Glhl (Athwal and Pathak, 1972).

317

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TABLE XV Some Green Leafhopper-Resistant Varieties and the Resistance Genes Possessed by Them Variety

Resistance gene

Reference

Pankhari 203 ASD 7 IR8 Ptb 18 Jhingasail Godalki Lien-tsan Palasithari 601 Betong DNJ 97

Glh 1 Glh 2 Glh 3 Glh 1 + ?a Glh I Glh 2 Glh 2 Glh 2 Glh 3 Glh 3 Glh 3 glh 4 GZh 5

Athwal et al. (1971) Athwal e f al. (1971) Athwal et al. (1971) Athwal and Pathak (1972) Siwi and Khush (1977) Siwi and Khush (1977) Siwi and Khush (1977) Siwi and Khush (1977) Siwi and Khush (1977) Siwi and Khush (1977) Siwi and Khush (1977) Siwi and Khush (1977) Siwi and Khush (1977)

H5 Ptb 8 ASD 8

“Ptb 18 has two genes for resistance. One of them is allelic to Glhl but the allelic relationship of the second is not known.

The inheritance of resistance was investigated in 13 more varieties and was found to be under monogenic control (Siwi and Khush, 1977). Variety Jhingasail has Glhl for resistance. Glh2 conveys resistance in varieties Godalki, Lien-tsan 50, and Palasithari 601. Resistance in Betong, DNJ97, and H5 is conditioned by Glh3. Single dominant genes, which are independent of Glhl, GZh2, and CZh3, convey resistance in ASD8, ARC6602, DM77, DSl ,and Khama 49/8. A single recessive gene, which is independent of Glhl, Glh2, and GEh3, conveys resistance in Ptb8; it was designated gZh4. The dominant resistance gene of ASD8 was designated GZh.5. The allelic relationships of the dominant genes conveying resistance in ARC6602, DM77, DS1, and Khama 49/8 t o each other and to glh4 and Glh5 are not known. Further investigations are in progress. The resistant varieties, which have been analyzed genetically to date, and their genes for resistance are listed in Table XV. d. Breeding for Resistance. Several parents-Peta, FB24, Tjeremas, and Sigadis-that were used in the crossing program at IRRI in the earlier years were found resistant to green leafhopper later on (Cheng and Pathak, 1972). IR8 and IR5 inherited resistance from Peta, and IR24 from Sigadis. Breeding line IR26243-8-11 (Peta3 /TN1), selected for good plant type, is also resistant to green leafhopper. IR8, IR262-43, and IR24 were used as good plant-type parents in many crosses at IRRI and in the national breeding programs. Thus, when we started screening the breeding materials for resistance to green leafhopper in 1969, a large number of our elite breeding lines were found resistant. Most of the crosses made at IRRI after 1969 had at least one parent with resistance to

318

GURDEV S. KHUSH

the insect. Progenies of those crosses were regularly evaluated and only those showing resistance were saved. Thus, over 90% of the entries included in the replicated yield trials at IRRI in 1973 and 1974 were resistant. All the IRRI named varieties, except IR22, as well as a great majority of breeding lines listed in Tables 111, V, VII, VIII, and XI11 are resistant to green leafhopper. Several varieties developed by the national programs such as Pelita I / l , Bahagia, C4-63, TN73-1, RDl to 5, Chandina, Biplab, Jaya, Pankaj, and Jayanti are also resistant. The resistance of varieties IR5, IR8, IR20, IR24, IR26, and IR30 is conditioned by GZh3. IR32 probably has Glhl for resistance. IR28, IR29, and IR34 inherit their resistance from Gam Pai 15. However, the allelic relationship of the resistance gene of those varieties is not known. Most resistant varieties developed in other programs have Glh3 for resistance. Most breeding lines at IRRI also have GZh3 for resistance. However, the proportion of materials having Glhl or GZh2 is increasing rapidly. Newly identified genes gZh4 and Glh5 are being incorporated into the improved plant-type background.

2. Green Rice Leafhopper The green rice leafhopper Nephotettix cincticeps Uhler occurs in Taiwan, Korea, Japan, and China (Fig. 3) and does considerable damage to the rice crop in those countries. According to Yoshimeki (1967), a total of 240,000 tons of rice were destroyed in Japan in the leafhopper outbreak of 1940. The green rice leafhopper also transmits the rice dwarf and yellow dwarf virus diseases. Biotype variation in the insect has not been reported. Ishii et aZ. (1969) found varieties Dharial, Kaladumi, Tadukan, and Tetep to be resistant t o the insect under field conditions in Japan. Murunga 137, Pannetti, and several other varieties were identified as resistant in Taiwan in greenhouse tests (Chow and Cheng, 1971). H105 and its progeny, IR4-93, are resistant in Korea (Table XIV). Inheritance of resistance has not been studied. Breeding work on resistance is being done in Taiwan and Korea.

D. GALLMIDGE

The rice gall midge Pachydiplosis olyzae Wood-Mason is the most serious rice pest in several areas of Asia and Africa. It occurs in Indonesia, Malaysia, Thailand, Cambodia, Laos, Vietnam, Southern China, Burma, Bangladesh, Nepal, India, Sri Lanka, and several African countries such as Sudan, Cameroons, and Nigeria. The female insect lays eggs near the base of the plant on the leaf ligules or in their vicinity on the leaf blade or leaf sheath. The newly hatched larvae creep down the leaf sheath to the browing points of the tillers and reach the

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319

interior of the shoot, where they feed on the meristematic tissues. Their feeding stimulates the tillers to grow into tubular galls which resemble onion leaf. The galls dry off without bearing any panicles. Early infestation results in profuse tillering, but the new tillers often become infested. Under heavy infestation, the crop appears badly stunted and very few panicles are produced. a. Biotype Variation. The possibility that biotypes of the insect exist was first pointed out by Shastry et al. (1972a). Their prediction has been confirmed by the differential reaction of several varieties in different rice-growing areas. Selections RP8 and RP9, developed from the crosses between IR8 and W1257 and IR8 and W1251, respectively, at Hyderabad, show high degrees of resistance at several locations in Andhra Pradesh and Madhya Pradesh, but are susceptible at the test locations in Orissa state in India. Similarly, the improved-plant-type variety Kakatiya is highly resistant in Warangal (Andhra Pradesh) but highly susceptible at Cuttack and Sambalpur (Orissa). Selections from the cross between IR8 and Siam 29 (RPW6 selections) are resistant in all those locations (D. V. Seshu, personal communication). The international gall midge screening tests have shown the existence of biotypic variation from one country to another. Selections from the crosses of Siam 29 and Ptb 18, which are resistant at all locations in India, are resistant in Sri Lanka but susceptible in Thailand and Indonesia. Conversely, RP8 and RP9 selections are resistant in Thailand but susceptible in Sri Lanka (S. V. S. Shastry, personal communication). Future international tests should throw more light on the distribution of different biotypes in different countries. b. Varieral Resistance. Varietal differences in susceptibility were recorded by Hegdekatti (1927), Li and Chiu (1951), Ramiah and Rao (1953), Khan and Murthy (1955), and Sen (1957). The differences were of quantitative nature. Bhat et al. (1958) identified the first highly resistant variety. They reported that Ratnachudi had less than 1% galls under severe field infestation, while all other test varieties had more than 25%. Ou and Kanjanasoon (1961) found Muey Nawng to be highly resistant in Lampang province of Thailand. Five varietiesW-791, W-352-A, W-613, W-676, and W-744-were resistant (Israel et ab, 1963) at CRRI. Kovitvadhi (1963) identified four resistant varieties from preliminary trials at four places in Thailand. A large number of varieties from various sources, totaling 5960, were screened at CRRI under field conditions during the monsoon seasons, between 1950 and 1970. Several varieties with varying degrees of resistance were identified; Ptb I8 and Ptb 21 from Kerala state of India and Leaung 152 from Thailand were found highly resistant (CRRI, 1970). Field tests at Warangal Station (Andhra Pradesh) between 1954 and 1964 showed Eswarakora, HR42, HR63, and Siam 29 as highly resistant. The resistance of Ptb 18 and Ptb 21 was also confirmed. Between 1968 and 1970, a large number of local as well as introduced varieties were screened by AICRIP under field conditions at Warangal station. These tests

320

GURDEV S. KHUSH

confirmed the resistance of F’tb 18, Ptb 21, Eswarakora, Siam 29, JBS446 (Desibayahunda), and JBS673 (Ratnachudi). In addition, 44 out of 867 ARC varieties tested in 1969 were found resistant (Shastry et al., 1971, 1972a). Field tests at CRRI showed the outstanding resistance of Ptb 10, Ptb 27, Ptb 28, F’tb 32, AC355, AC1368, Peykeo P.129, and Peykeo E.53 (P. S . Prakasarao, personal communication). Some of the resistant varieties are listed in Table XVI. c. Inheritance of Resistance. The inheritance of resistance to gall midge was studied by K. V. L. Narashima Rao at AICRIP. Segregating populations from the crosses IR8/W.1263 and IR8/Ptb 21 were screened under heavy insect pressures at Warangal station. Two and four genes for resistance were postulated in W1263 and R b 18, respectively (Shastry et al., 1972a). Sastry and Prakasa Rao (1973) investigated the inheritance of resistance in W1263 and W12708 at CRRI and postulated the existence of three recessive genes for resistance. In a more diagnostic study on the inheritance of resistance, Satyanarayanaiah and Reddi (1972) postulated the existence of one dominant gene for resistance in W. C. 1263. d. Breeding for Resistance. By far the greatest amount of work on breeding for resistance has been done in India. Breeding programs aimed at combining gall midge resistance with other desirable traits were initiated at CRRI and at Warangal station even before the advent of the improved plant type. At CRRI, several resistant selections with tall stature (CR55 and CR56 selections) came from crosses involving resistant parents R b 18 and Ptb 21. At Warangal, semicommercial varieties W1251, W1253, W1257, and W1263 were developed from the cross between MTU15 and Eswarakora. After the introduction of the dwarf plant type, attempts to combine gall midge resistance with improved plant type were made through extensive hybridization programs at AICRIP, CRRI, and Warangal. Several resistant parents were crossed with IR8. Improved plant-type selections from those crosses were evaluated for resistance under field conditions at the respective research centers. Promising resistant selections from all the programs were then screened and tested for yield in coordinated trials at several locations where gall midge is endemic. Several resistant selections of improved plant type were identified from those tests and retested in minikit trials in farmers’ fields as well as in the international gall midge screening tests. Some selections have been released as varieties in different states of the country. RPW6-13 and W13400 from the cross between IR8 and Siam 29 were released for cultivation in Karnatka and Andhra Pradesh, respectively. Kakatiya, a resistant variety from the cross IR8/W1263, was recommended in Andhra Pradesh. CR93-4-2, a promising selection from the cross Ptb 21/Ptb 18//IR8, was approved for cultivation in Orissa under the name Sakti. Several other resistant selections-RP9 selections from the cross of IR8 and W1251, and CR94MR-1550 selection from the cross of Ptb 21/Ptb 18//IR8have shown promise in several areas of the country.

32 1

DISEASE AND INSECT RESISTANCE IN RICE

At AICRIP, intercrosses between the F1 hybrids have been made to combine resistance from two donor parents. RP35 1 selections from the cross IR8/Siam 29//Eswarakora/IR8 as well as RP352 selections from the cross IR8/Ptb 18//Eswarakora/IR8 have shown resistance at all test locations in India, and presumably combine resistance genes from different donors together. Resistant germ plasm from India was introduced into Thailand in 1967, and many multiple crosses were made between local varieties, improved plant-type varieties and gall-midge-resistant varieties, such as W1240, W1252, W1256, W1259, and W1263. Improved plant-type selections from these crosses were evaluated for resistance to gall midge under field conditions, and many resistant progenies were identified (Pongprasert et al., 1972). The resistant variety RD4 was released in 1973. Several more promising selections with resistance are under consideration for varietal release. In Sri Lanka, gall-midge-resistant tall donors as well as resistant breeding lines from India are utilized as sources of resistance in the breeding programs. W1263 was crossed with several improved plant-type, susceptible varieties such as LD66, IR8, BG11-11, and BG35-5. The hybrid progenies were backcrossed three or four times, with improved plant-type varieties as recurrent parents. Several resistant selections with good attributes of the recurrent parents have been selected and tested for yield in the country. Two AICRIP selections with improved plant type from the cross IR8/F’tb 18//Eswarakora/IR8 (RP352) were highly resistant in Sri Lanka. They were extensively used in single, three-way, and double crosses as well as in backcrosses with other improved plant-type varieties. Promising resistant selections from these crosses are being evaluated in yield trials. TABLE XVI Some Varieties Showing Resistance to Gall Midge in Different Countries ~~

~

~

~

India (CRRI)

India (Warangal)

Thailand

Indonesia

Ptb 10 Ptb 18 Ptb 21 Ptb 27 Ptb 28 Ptb 3 2 Leuang 152 AC 355 AC 1368 Peykeo P129 Peykeo E53 ARC 6202 ARC 7317

Eswarakora HR 4 2 HR 6 3 Siam 29 JBS 446 JBS 673 Ptb 10 Ptb 18 Ptb 21 ARC 6221 ARC 10494 ARC 109322 ARC 10817

Muey Nawng 62M Muey Nawng 6 2 Muey Nawng Fang Dok Putdor Eswarakora W 1251 W 1253 W 1257 W 1263

Leri Gundul Gundul Sempol Gama 6 1 Jimbruk ARC 10817 ARC 10932-2 ARC 105 20 ARC 10494 Siam 29 Eswarakora W 1263 Muey Nahng 62M

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GURDEV S. KHUSH

Several gall-midge-resistant breeding lines from India and Thailand, and native resistant varieties are being used as sources of resistance to gall midge in the breeding program in Indonesia. Breeding lines from the crosses involving resistant parents are screened under field conditions in West Java. At IRRI, Ptb 18 and CR93-14 (a breeding line from CRRI) have been used as sources of resistance t o gall midge. Improved plant-type selections with multiple resistance to other diseases and insects were screened for gall midge reaction in cooperation with CRRI scientists. Several promising selections with high level of resistance to gall midge were identified. One of them, IR2070-747-6-3, was named IR32 in 1975. Two other resistant selections, IR2071-625-3-252 and IR2070-423-2-5-6,were named IR36 and IR38 in 1976 in the Philippines. I V . Developing Varieties with Multiple Resistance

In most rice-growing areas serious yield losses are caused by more than one disease or insect. In Latin America, for example, blast, hoja blanca, and Sogatodes oryzicola are the limiting factors to rice production. In Africa, blast and stem borers seriously reduce rice yields. In Asia where 92% of the rice is produced and consumed, more than a dozen diseases and insects cause losses of epidemic proportions. In tropical Asia, the disease and insect problem is most serious because of year-round favorable climate and long history of rice cultivation with consequent evolution of many disease and pest organisms. One year there may be an epidemic of bacterial blight, the next year green leafhopper and tungro may cause serious damage, and the following year an outbreak of brown plant hopper and grassy stunt might occur. To minimize yield losses from disease and insect attacks, varieties with multiple resistance to most major diseases and insects are required. Five diseasesblast, sheath blight, bacterial blight, tungro, and grassy stunt-and four insectsbrown plant hopper, green leafhopper, stem borers, and gall midge-commonly occur in most countries of tropical Asia. Development of improved plant-type varieties with multiple resistance to those diseases and insects has been the major objective of the IRRI breeding program for several years. Breeding for resistance to blast and bacterial blight began in 1965, to tungro and stem borers in 1966, to brown plant hopper and green leafhopper in 1968, to grassy stunt in 1969, to gall midge in 1971, and to sheath blight in 1974. Because of an anticipated increase in the incidence of white-backed plant hopper, breeding work for resistance to that insect was initiated in 1975. Rapid progress has been made in developing improved germ plasm with resistance to blast, bacterial blight, tungro, grassy stunt, brown plant hopper, and green leafhopper. Only 12%of the entries in replicated yield trials planted in the wet season of 1969 were resistant to blast (Fig. 4). The proportion rose to

DISEASE AND INSECT RESISTANCE IN RICE

0

m 1969 70

1969 7 0 71 72 7 3 74

323

71 72 73 74

FIG. 4. Change in the proportion of F, populations and entries in the replicated yield trials at IRRI with resistance to important insects and diseases (Khush, 1977). Reproduced by permission of the New York Academy of Sciences.

60% in the wet season of 1974. Similarly, only 17% of the entries in replicated yield trials of 1969 wet season were resistant to bacterial blight. The proportion increased to 98% in the 1974 wet season (Fig. 4). Progress with tungro, grassy stunt, brown plant hopper, and green leafhopper has been equally dramatic (Fig. 4). The main thrust of the program, of course, has been to combine resistance to all those diseases and insects with improved plant type. Equally rapid progress has been made in this direction. About 87% of the entries in the replicated yield trials of the 1969 wet season were either susceptible to all six diseases and insects (blast, bacterial blight, tungro, grassy stunt, brown plant hopper, and green leafhopper) or resistant to only one of them (Fig. 5). Only 2% of the entries were resistant to three diseases and insects. The proportion of entries with multiple resistance gradually increased, and in the 1974 trials, 90% were resistant either to five diseases and insects or to all six. Six of the multiple

324

GURDEV S. KHUSH Resistonce to a wrnber of d i m s and insects (%)

FIG. 5. Change in proportion of entries in annual replicated yield trials with multiple resistance to important diseases and insects (blast, bacterial blight, tungro, grassy stunt, brown plant hopper, and green leafhopper) at IRRI. Each year’s trial consisted of about 185 entries (Khush, 1977). Reproduced by permission of the New York Academy of Sciences.

resistant lines were named varieties and recommended for cultivation in several countries. Table XVII shows the disease and insect reactions of all IRRI named varieties and indicates the progressive increase in the levels of resistance of the newer rice varieties. The breakthrough in developing the improved plant-type germ plasm with multiple resistance to major diseases and insects was achieved through a well-planned breeding program and through a liberal exchange of ideas and materials between IRRI scientists and rice scientists in other rice-growing countries. Salient features of the breeding methodology and procedures employed and international cooperation are discussed in the following section.

A. BREEDING METHODS AND PROCEDURES

I . Choice of Parents

A major strength of the breeding program at IRRI has been the well-stocked germ plasm bank. From 256 accessions in 1961, the germ plasm collection

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325

increased to 6900 entries a year later, and to 23,560 entries in 1972 (Chang et al., 1975). It now has about 40,000 accessions from 73 countries of the world. Another major strength has been the presence of highly competent pathologists and entomologists on the Institute staff. As soon as a serious disease or insect problem was identified, scientists started developing screening techniques and evaluating germ plasm for resistance. The identified sources of resistance were immediately introduced into the crossing program. The donor parents generally had poor plant type characterized by tall stature, droppy leaves, weak stems, and consequently, low-yield potential. The first step therefore was to transfer those sources of resistance to an improved plant type (short stature, erect leaves, sturdy stems, high tillering). This “conversion” was achieved by crossing the donor parents with an improved plant-type parent, growing large Fz populations (2000-5000 plants), and selecting improved plant-type segregates. The selected plants were examined for grain quality, and F3 progenies were grown from those with good grain quality. The F3 progenies were evaluated for the resistance trait under study, and several resistant selections (generally up to 100) with good grain quality, improved plant type, and appropriate growth duration were saved for further evaluation in the F4 and F5 generations. Through repeated evaluations, a few (2-10) true-breeding, improved plant-type selections with resistance to given traits were selected. Between 1965 and 1969, IR8, several selections of IR262 (IR262-43-8 in particular), and IR24 were used as improved plant-type parents. Several donor parents were used for each disease and insect. 2. Crossing Program

During the period when emphasis was on germ plasm conversion, single crosses were made. Poor progenies were obtained in a multiple cross if more than one parent had poor plant type. Since very few improved plant-type parents were available in the initial stages, only single crosses were feasible. About 200 to 400 such crosses were made each year. By the end of 1970 a large number of improved plant-type breeding lines with resistance to one or two diseases and insects became available. To combine resistance to all the major diseases and insects together, the crossing program was expanded and a large number of multiple crosses were made employing those breeding lines. A large number of single crosses between those lines were made each season. The following season either the two F l s were crossed with each other or an Ft was crossed with a third breeding line. A fairly large number of F, seeds (300-400) were obtained from multiple crosses. This allowed the gametic variability of single-cross F, s to be sampled. In producing single-cross F, s, each breeding line was crossed with a number of other breeding lines. Thus, a set of single-cross F1 progenies were available for making double crosses or topcrosses in the next season, and appropriate combinations could be selected t o combine the resistance to given diseases and insects.

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GURDEV S. KHUSH

That also allowed the rapid determination of the combining ability of the breeding lines. A breeding line that yielded poor progenies in a number of cross combinations was assumed to be a poor combiner and was removed from the crossing program. 3. Handling Segregating Populations The pedigree method of breeding was employed almost exclusively in handling the segregating materials. Selection work was based on comprehensive records on the disease and insect reactions of each line and, in the case of F4 and advanced generation lines, on the reaction of the ancestral lines as well. The bulk method of breeding was not used because it does not permit concurrent screening for a number of diseases and insects. The backcross method was not used for lack of suitable recurrent parents. A few backcrosses were made in the crosses with Oryza nivara for the program on resistance to grassy stunt. IR8, IR20, and IR24 were used as recurrent parents. After 3 to 4 backcrosses, we obtained breeding lines similar to IR8, IR20, or IR24, but they lacked resistance to other important diseases and insects, such as tungro and brown plant hopper, and were again entered in the crossing program. Now varieties and breeding lines with multiple resistance are available, and we are using the backcrossing program to incorporate resistance to white-backed plant hopper. For traits under polygenic control, the pedigree method is not as suitable. At IRRI, the diallele selective mating system, originally suggested by Jensen (1970), is being tried for combining minor genes for resistance to stem borers and whorl maggot from several sources. This method involves: (1) crossing a number of moderately resistant parents (generally 5-6) in all possible combinations, (2) intercrossing the F1s so obtained in all possible combinations, ( 3 ) screening the double-cross F1 progenies for resistance, and (4) intercrossing the selected plants that have better resistance than either parent. This crossing, screening, and selection process is continued until the minor genes from different sources are accumulated and the intensity of the trait is built up. We are in the third cycle of this type of recurrent selection program for stem borer resistance and the second cycle of selection for the whorl maggot program. The success of the method is difficult to prognosticate at this stage.

4. Screening Segregating Populations The success of the disease- and insect-resistance breeding program depends to a large measure upon the fidelity, speed, and practicability of the screening technique. Various greenhouse and field screening methods employed at iRRI have been tailored to accommodate large volumes of breeding materials. About 50,000 pedigree rows are grown each year at IRRI. Most are screened for

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327

reaction to all the major diseases and insects. If possible, the breeding materials are exposed t o artificially created or naturally occurring disease and insect epiphytotics. Previously, breeding nurseries were grown with full insecticide protection; however, since 1970 most of the nurseries are grown without insecticide treatment to allow a buildup of huge populations of plant hopper, leafhopper, and stem borers. Rant hoppers and leafhoppers in large numbers also insure the spread of virus diseases in the nurseries. Sometimes artificially virusinoculated plants are planted around the borders of nurseries t o provide a source of inoculum. The insect populations are manipulated by applying selective insecticides. At IRRI farm, Diazinon has no toxic effect on the brown plant hopper but it kills all predators and other natural enemies of t h s insect. By judicious application of Diazinon, an outbreak of brown plant hopper has been induced in the IRRI nurseries. This has also led to an increased incidence of grassy stunt. All nurseries are artificially inoculated with bacterial blight in the field and tested for reaction to blast in the blast nurseries. Data on green leafhopper and brown plant hopper reactions are also obtained from greenhouse tests. Selected materials are planted at other locations in the Philippines under disease and insect pressures different from those at IRRI. Every effort is made to eliminate the susceptible materials in the early generations. Screening begins in the F1 generation of multiple crosses. For example, consider a double cross between four parents; A is resistant to bacterial blight, B is resistant t o grassy stunt, C is resistant to brown plant hopper, and D is resistant to green leafhopper. All these traits are controlled by single dominant genes, whch segregate independently of each other. About 400 seeds from the double cross A/B//C/D are obtained. The most logical system for screening the progenies would be to germinate and inoculate all 400 seedlings with grassy stunt in the greenhouse. Half of the seedlings would be susceptible and will be eliminated. The remaining 200 would be transplanted in the field and inoculated with bacterial blight; half of the 200 that would be susceptible would be rogued out. Seeds from the remaining 100 plants would be harvested individually. Two small seed samples would be taken out from each and the progeny tested for resistance to brown plant hopper and green leafhopper. Those carrying the brown plant hopper resistance gene (50%) and the ones carrying the green leat3opper resistance gene (50%)would be identified. F2 populations would be grown only from those carrying both genes (25-30 plants). Thus, by judicious and timely screening, the original F, sample of 400 would be reduced to 25 to 30 plants, and F2 populations would be grown from these plants. All these F2 populations would be segregating for the four resistance genes. They could be subjected to appropriate disease and insect pressures. Agronomically desirable plants with multiple resistance would be selected and rescreened in the F, and F4 generations to obtain true breeding lines.

328

GURDEV S. KHUSN

A large number of multiple crosses between breeding lines of improved plant type, known combining ability, and resistance to a number of diseases and insects are made each season and screened according to the outlined procedure. New parents with different sources of resistance are constantly included in the crossing program. This integrated varietal development program has resulted in superior germ plasm with resistance to all major diseases and insects. Newer lines with different genes and gene combinations for resistance should continue to come from the program.

B. INTERNATIONAL COOPERATION

International and interdisciplinary cooperation has been the key ingredient of the varietal development program at IRRI. Liberal exchange of ideas and materials between different programs, cooperative testing for disease and insect resistance in the Philippines at the Bureau of Plant Industry Stations, and in several other countries such as India, Sri Lanka, Bangladesh, Thailand, and Indonesia has contributed greatly to the development of germ plasm that is resistant to diseases and insects. The pedigree of IR28, IR29,and IR34 (Fig. 6 ) illustrates this international and interdisciplinary approach. To develop these high-yielding, good grain quality, and multiple disease- and insect-resistant varieties, eight varieties from six different countries were used in the crossing program. The seeds of these varieties and 40,000 others were supplied by scientists from those countries. This germ plasm was evaluated by pathologists and entomologists for disease and insect resis-

1

I

IR1561 BB BPH

1 IRE33 BL T GLH

IRR37 0s

GLH

I IR2M2 EL BEGS BPH QLH

n IRL0,IRLoBIRW BL EBT GS BPH Wli

FIG. 6. Pedigree of IR28, IR29, and IR34. The progress in combining together the resistance to six major diseases and insects from several parents is indicated. BL = Blast disease; BB = bacterial blight disease; T = tungro virus disease; GS = grassy stunt virus disease; BPH = brown plant hopper; and GLH = green leafhopper (Khush, 1977). Repre duced by permission of the New York Academy of Sciences.

329

DISEASE A N D INSECT RESISTANCE IN RICE

tance, respectively. The breeders combined the identified sources of resistance to diseases and insects with improved plant type. The segregating populations were tested at IRRI and the Philippine Bureau of Plant Industry Station at Maligaya, and in Indonesia. The seeds of the improved germ plasm as well as of the entries in the germ plasm bank are shared with scientists all over the world. Up to the end of 1975 more than 95,000 seed samples of breeding lines were supplied to requesting parties in 80 countries of the world. The breeding lines are used as parents in the crossing programs, and some have become named varieties. To date 40 breeding lines from IRRI have been named varieties in other countries. The recently expanded international testing program will facilitate the exchange and dissemination of germ plasm between the various rice improvement programs.

V. Stability of Resistance

There is growing support for the contention that the resistance governed by polygenes-also referred to as general resistance or horizontal resistance-is more lasting than resistance governed by major genes (specific or vertical resistance). When the program on breeding for disease and insect resistance in rice was initiated at IRRI, little was known about the genetics of resistance. Available donor parents were used as sources of resistance and we developed the improved plant-type breeding lines and varieties with multiple resistance to as many as four diseases and four insects (Table XVII) within a short period of 7 to 8 years. TABLE XVII Disease and Insect Resistance Reactions of IRRI Named Varieties Disease and insect reaction'

Variety

Blast

Bacterial bIight

IR5 IR8 IR20 IR22 IR24 IR26 IR28 IR29 IR30 IR32 IR34

MR S MR S S MR R R MS MR R

S S R R S R R R R R R

Grassy stunt S S

S S S MS R R R R R

Tungro

Green leafhopper

Brown plant hopper

Stem borer

Gall midge

S S MR S S MR R R MR MR R

R R R S R R R R R R R

S

MS S MR S S MR MR MR MR MR MR

S S S S S S S S S R S

S S S S R R R R R R

'S = Susceptible; MS = moderately susceptible; MR = moderately resistant; R = resistant.

330

GURDEV S . KHUSH

During this period at IRRI, by investigating the mode of inheritance of resistance to some diseases and insects, it was found that resistance to most diseases and insects with the exception of stem borer, is controlled by the major gene.

A. VERTICAL RESISTANCE

Information on the stability of vertical or major gene resistance in rice is meager. As discussed earlier, bacterial blight-resistant IR20 and IR26, which have Xa4, have been widely grown in the tropics. Their resistance has held up except in a small area of the Philippines where a strain of bacterium that is moderately virulent to Xa4 has appeared. This strain has remained localized and causes only slight damage to rice varieties with Xa4. Several bacterial blight-resistant varieties, such as Benong, Sigadis, Syntha, and Dewi Tara, have grown in Indonesia for 10 t o 20 years. The resistant TKM6, MTU15, and CO 21 have grown in India for many years. The occurrence of bacterial strains virulent to varieties with host resistance has not been reported. Several varieties resistant to green leafhoppers-Peta, Intan, and Bengawanwere widely grown in Indonesia and the Philippines for 30 to 35 years. Several improved-plant-type varieties-IR5, IR8, IR20, IR26, and C4-63, which inherited GZh3 for resistance to green leafhopper from Peta-have also been grown for several years. No clear-cut evidence for the origin of green leafhopper biotypes that are virulent to GZh3, under the influence of host resistance has been found. However, varieties resistant t o brown plant hopper became susceptible within 1.5 years of their introduction into the British Solomon Islands, because of the appearance of a new brown plant hopper biotype. Similarly, within 2 years of its large-scale cultivation in the Philippines, IR26 was attacked by new biotypes of the insect in several localities. The germ plasm for resistance to brown plant hopper comes from South India and Sri Lanka where biotypes of the insect are virulent t o those varieties. These biotypes probably originated under the influence of host resistance. The influence of host resistance on the insect populations of brown plant hopper and green leafhopper is obviously different. The difference may be due to the differential selection pressure exerted by the resistant varieties on insect populations. The level of resistance to brown plant hopper conveyed by Bphl and bphZ is sufficiently high that the insect cannot perpetuate itself on resistant varieties. It either changes or is eliminated. On the other hand, the level of resistance to green leafhopper conditioned by GZh3 is only moderate. The insect feeds on resistant plants and reproduces, although at a much lower rate than it can when feeding on susceptible varieties. Thus, it can perpetuate itself on resistant varieties but chances for the origin of more virulent biotypes are considerably lower than those for the brown plant hopper. Thus, the useful life

DISEASE AND INSECT RESISTANCE IN RICE

33 1

of the Glh3 gene may be considerably longer than that of either Bphl or Bph2. The other genes for resistance to the green leafhopper-Glhl and Glh2-convey higher levels of resistance comparable with those of Bphl for brown plant hopper. When varieties having either gene are grown widely, new biotypes of the green leafhopper might arise rapidly. Differences in the inherent capacity of the two insect species to change under the influence of host resistance may also be responsible for the differences in longevity of resistance to the two insects. The strategy at IRRI to utilize major gene resistance in rice is twofold. The short-term strategy aims at incorporating the known major genes for resistance to different diseases and insects into the improved plant-type background, combining these genes in different combinations, and sharing the resulting germ plasm with other programs. IRRI is close to meeting this goal. The long-term objective is t o identify several genes for resistance to each disease and insect, particularly bacterial blight and the brown plant hopper. As soon as a new gene is identified, it is transferred to an improved plant-type background. When a number of genes become available it would be possible to adopt any of the following approaches to utilize these genes for vertical resistance: 1. Release one gene for resistance and wait until it becomes ineffective; release the second gene, and so on. This approach was adopted to control stem rust of wheat in Australia between 1938 and 1950 (Watson and Luig, 1963). This approach is being taken with respect t o brown plant hopper resistance. IR26 was released during the brown plant hopper outbreak of 1973 in the Philippines. This variety and IRI 561-228-3, another brown plant-hopper-resistant selection, were grown widely in the Philippines in 1974 and 1975. Both cultivars have Bphl for resistance. Toward the end of 1975 and in 1976, hopperburn on these varieties was reported in two locations in the Philippines. IR36 and IR38 which have bph2 for resistance to brown plant hopper, were hastily released by the Philippine Government in March 1976. IR36 and IR38 are expected to hold for a couple of years. By that time varieties with Bph3 and bph4 would be available. 2. Pyramid two, three, or even more major genes together in the same variety, as suggested by Watson and Singh (1952). Several wheat varieties that combined up to five genes for resistance to stem rust were developed. Canadian breeders have adopted the same procedure for developing oats that are resistant to crown rust (Knott, 1974). Several scientists, most notably Nelson (1972), favor this approach. This approach depends upon the existence and availability of several races or biotypes capable of distinguishing between genotypes with various numbers of resistance genes. Otherwise the breeding procedure becomes too lengthy. The availability of several biotypes of brown plant hopper makes it feasible to pyramid genes for resistance to this insect. 3. Develop multiline cultivars, as proposed by Jensen (1952) and Borlaug (1958). This approach was followed for crown rust resistance in oats in Iowa (Browning et al., 1969), and a program to develop multiline stem-rust-resistant

332

GURDEV S. KHUSH

cultivars of wheat is under way at Centro Internacional de Mejoramiento de Maiz y Trigo. The development of multilines involves an extensive program of gene identification and backcrossing. As suggested in an earlier section, this approach merits serious consideration for an international project on blast. 4. Develop resistant varieties with different resistance genes and recommend them for different geographical regions of the country where the crop covers a sizable area. As pointed out by Nelson (1972), this type of gene deployment is essentially a geographical multiline. A formal plan for regional deployment of genes is in effect for resistance t o crown rust in oats in Iowa (Frey et al., 1973). This approach may be followed for either rice diseases or insects when enough genes are identified.

B. HORIZONTAL RESISTANCE

At IRRI the search for horizontal or polygenic resistance in rice continues. The Institute program on stem borer resistance deals with polygenic systems. Polygenic variation has been noted for the resistance to brown plant hopper, grassy stunt, and bacterial blight. However, there are practical difficulties in exploiting this variation. One concerns the breeding system of the crop. In an outcrossing species a number of cultivars, with minor genes that are desirable for accumulation, can be mixed, planted, and allowed to interbreed for several generations. Appropriate disease or insect pressure is applied to each generation, and individuals with higher levels of resistance are selected for growing the next generation. Random mating permits the formation of new gene combinations at each generation, and recurrent selection changes the gene frequency for higher levels of resistance. The process cannot be followed with rice because of its self-pollinating nature. However, a usable source of male sterility that can be employed for inducing high rates of outcrossing in a composite population with several sources of polygenic resistance is being sought. Pending the availability of a male sterile, the diallele selective mating system discussed in an earlier section is being used. The second difficulty concerns the screening techniques. Most artificial screening techniques fail to detect polygenic differences. During the brown plant hopper outbreak of 1973 at the IRRI farm, several selections with tolerance to the insect were identified. They withstood the insect attack longer than the susceptible varieties did, but were eventually killed. However, when they were tested in the greenhouse, these differences could not be detected. At IRRI, a breeding program on horizontal resistance to brown plant hopper was initiated, using these sources and following the diallele selective mating system. When the F1 progenies from the double crosses were ready for testing, there were no brown plant hoppers in the field.

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333

It is important to develop horizontal resistance to the brown plant hopper, and efforts are being made at IRRI to screen the breeding population in other countries, such as the British Solomon Islands, where field populations of the brown plant hopper are always high.

VI. Conclusions

Among cereal crops, rice is the host of the largest number of diseases and insect pests. These cause serious yield losses annually. The magnitude of losses caused by the diseases and insects is likely to increase as the level of rice production per unit area increases. The germ plasm resources for disease and insect resistance are vast, but only a portion has been collected and evaluated for resistance. Much germ plasm remains to be collected and catalogued. It should be collected before it becomes extinct through the adoption of high-yielding varieties. The germ plasm that has not been evaluated, especially the national germ plasm collections, should be evaluated to identify more sources of resistance. Different sources of resistance should be genetically analyzed to identify diverse genes for resistance. A systematic international survey of races or biotypes of major diseases and insects should be carried out with the use of differential varieties. Sources of resistance to all races or biotypes should be identified and genetically analyzed. The different major genes for resistance should be utilized according to needs of each program. Various alternatives are discussed. Greater efforts should be expended in studying and utilizing horizontal resistance, although vertical resistance will continue to be useful for years to come. Interdisciplinary cooperation among the pathologists, entomologists, and breeders is essential for the speedy implementation of host resistance progcams. International cooperation is essential in collecting and evaluating germ plasm, studying the races and biotypes, identifying the diverse genes for resistance, cooperative testing for disease and insect resistance, and liberal exchange of improved germ plasm.

REFERENCES Abeygunawardena, D. V. W. 1967. Proc. Symp. Rice Dis. Their Control Growing Resistant Varieties Other Measures pp. 171-179. Agric., For. Fish. Res. Counc., Tokyo. Abeygunawardena, D. V. W., Bandaranayaka, C. M., and Karandawela, C. B. 1970. Trop. A@. (Ceylonj 126, 1-13.

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Sakurai, Y. 1969. In “The Virus Diseases of the Rice Plant,” pp. 257-275. Johns Hopkins Press, Baltimore, Maryland. Sakurai, Y., and Ezuka, A. 1964. Chugoku Nogyo Shikenjo Hokoku A 10,Sl-70. Sakurai, Y., and Toriyama, K. 1967. Proc. Symp. Rice Dis. Their Control Growing Resistant Varieties Other Meas. pp. 123-135. Agric., For. Fish. Res. Counc., Tokyo. Sakurai, Y., Ezuka, A., and Okamoto, H. 1963. Chugoku Nogyo Shikenjo Hokoku A 9, 11 3-1 25. Samper, A. 1968. Bull. Entomol. Soc. Am. 14,128-130. Sasaki, R. 2922. J. Plant Prof. {Jpn.) 9,631-644. Sasaki, R. 1923. J. Plant Prof. (Jpn.) 10.1-10. Sastry, M. V. S., and Prakasa Rao, P. S. 1973. Curr. Sci. 42,652-653. Satyanarayanaiah, K., and Reddi, M. V. 1972. Andhra Agric. J. 19, 1-8. Seetharaman, R., Sharma, S. D., and Shastry, S. V. S. 1972. In “Rice Breeding,” pp. 187-200. Int. Rice Res. Inst. (IRRI), Los Baiios, Philippines. Seko, H., and Kato, I. 195O.Proc. Crop. Sci Soc. Jpn. 19(1/2), 201-203. Sen, A. C. 1957. Proc. Indian Sci. Congr., 44th Part 111, p. 402. Serrano, F. B. 1957. Philipp. J, Sci. 86, 203-230. Shastry, S. V. S., Sharma, S. D., John, V. T., and Krishniah, K. 1971. In?. Rice Commun. Newsl. 20(3), 1-16. Shastry, S. V. S., Freeman, W. H., Seshu, D. V., Israel, P., and Roy, J. K. 1972a. In “Rice Breeding,” pp. 353-365. Int. Rice Res. Inst. (IRRI), Los Baf~os,Philippines. Shastry, S . V. S., John, V. T., and Seshu, D. V. 1972b. In “Rice Breeding,” pp. 239-252. Int. Rice Res. Inst. (IRRI), Los Baflos, Philippines. Shekhawat, G. S., Srinivastava, D. N., and Rao, Y. P. 1972. Indian J. Agric. Sci. 42, 11-15. Shigemura, C., and Kitamura, E. 1954. Nogyo Gijutsu 9(3), 37-39. Shinkai, A. 1962. Bull. Natl. Inst. Agric. Sci., Ser. C 14, 1-112. Siang, W. N. 1952. Plant Dis. Rep. Suppl. 215,165-186. Singh, K. G. 1969. I n “The Virus Diseases of the Rice Plant,” pp. 75-78. Johns Hopkins Press, Baltimore, Maryland. Siwi, B. H., and Khush, G. S. 1977. Crop Sci. 17,17-20. Sonku, Y., and Sakurai, Y. 1967. Chogoku Nogyo Shikenjo Hokoku E 1,l-24. Suzuki, H., Kato, T., Kawaguchi, K., and Sasamura, H. 1960. Bull. Tochigipref: Agric. Exp. Stn. 4, 1-13. Takahashi, Y. 1965. In “The Rice Blast Disease,” pp. 303-329. Johns Hopkins Press, Baltimore, Maryland. Takahashi, Y. 1967. Proc. Symp. Rice Dis. Their Controt Growing Resistant Varieties Other Meas. pp. 157-170. Agric., For. Fish. Res. Counc., Tokyo. Tantera, D. M., Satomi, H.,and Roechan. 1973. Contr. Rex Inst. Agri. Bogor No. 2, 1-8. Tin Win, U. 1974. M.S. Thesis, Univ. of the Philippines, Coll. of Agric., Los Bafios, Philippines. Toan, T. H. 1969. I n “The Virus Diseases of the Rice Plant,” pp. 87-89. Johns Hopkins Press, Baltimore, Maryland. Tochinai, Y., and Sakamoto, M. 1937. J. Fac. Agric., Hokkaido Univ. 41, 1-96. Toriyama, K. 1965. Nogyo Oyobi Engei Agr. Hort. 40,641-644. Toriyama, K. 1969. In “The Virus Diseases of the Rice Plant,” pp. 313-334. Johns Hopkins Press, Baltimore, Maryland. Toriyama, K. 1972. In “Rice Breeding,” pp. 253-281. Int. Rice Res. Inst. (IRRI), Los Bafios, Philippines. Toriyama, K., Sakurai, Y., Washio, O., and Ezuka, A. 1966. Chugoku Nogyo Shikenjo Hokoku A 13.41-54.

DISEASE AND INSECT RESISTANCE IN RICE

34 1

Toriyama, K., Yunoki, T., and Shinoda, H. 1968a. Jpn. J. Breed. 18, Suppl. 1, 145-146. Abstr. Toriyama, K., Yunoki, T., Sakurai, Y., and Ezuka, A. 1968b. Jpn. J. Breed. 18, Suppl. 2, 157-158. Toriyama, K., Kariya, K., Washio, O., Sakamoto, S., Yamarnoto, T., and Shinoda, H. 1968c. Chugoku Nogyo Shikenjo Hokoku A 16,l-18. Tsutsui, K. 1951. Nogyo Cijutsu 6(2), 40-43. Tu, J. C. 1967. Plant Dis. Rep. 51,682-684. Tullis, E. C. 1937. Phytopathology 27, 1007-1008. Ujihara, M. 1960. Jpn. J. Breed. 10, 113-114. Abstr. Van Der Meulen, J. G. J. 1951. Contrib. Gen. Agric. Res. Sfn. 116, 1-38. Bogor. van Halteren, P., and Sama, S. 1974. Lembaga Penelitian Maros, Bull. 3. Vorrauri, X., and Giatgong, P. (197O).Ninfh Natl. Con$ Agr. Sci. (Feb., 1970), Bangkok. Washio, O., Kariya, K., and Toriyama, K. 1966. Chugoku Nogyo Shikenjo Hokoku A 13, 55-85. Washio, O., Toriyama, K., Ezuka, A., and Sakurai, Y. 1968a. Jpn. J. Breed. 8, 96-101. Washio, O., Toriyama, K., Ezuka, A., and Sakurai, Y. 1968b. Jpn. J. Breed. 18, 167-172. Washio, O., Ezuka, A., Toriyama, K., and Sakurai, Y. 1968c. Chugoku Nogyo Shikenjo HOkOkU A 16, 39-197. Wathanakul, L.. 1965. Natl. Conf. Agric. Biol., 4h, Kasetsart Univ., Bangkok. Wathanakul, L., and Weerapat, P. 1969. In “The Virus Diseases of the Rice Plant,” pp. 79-85. Johns Hopkins Press, Baltimore, Maryland. Wathanakul, K., Chainiangkol, U., and Kanjanasoon, P. 1968. FAO-IRC Working Party Rice Prod, Prot., 12th Meet., Peradeniya, Ceylon. Watson, I. A., and Singh, D. 1952. J. Aust. Inst. Agric. Sci. 18, 190-197. Watson, I. A., and Luig, N. H. 1963. Proc. Linn. SOC.N S . W. 38, 235-258. Yamaguchi, T., Yasuo, S., and Ishii, M. 1965. J. Cent. Agric. Exp. Stn. 8, 109-160. Yamasaki, Y., and Kiyosawa, S. 1966. Bull. Natl. Inst. Agric. Sci., Ser. D 14, 3 9 4 9 . Yokoo, M., and Kiyosawa, S . 1970. Jpn. J. Breed. 20, 129-132. Yoshimeki, M. (1967). In “The Major Insect Pests of the Rice Plant.” pp. 181-194. The Johns Hopkins Press, Baltimore, Maryland. Yunoki, T., Ezuka, A., Morinaka, T., Sakurai, Y., Shinoda, H., and Toriyama, K. 1970. Chugoku Nogyo Shikenjo Hokoku E 6, 21-41. Yunus, A., and Rotschild, G . H. L. 1967. In “The Major Insect Pests of the Rice Plant,” PP. 6 17-642. Johns Hopkins Press, Baltimore, Maryland.

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TRICKLE-DRIP IRRIGATION: PRINCIPLES AND APPLICATION TO SO1L-WATER MANAGEMENT Eshel Bresler Division of Soil Physics. Institute of Soils and Water. Agricultural Research Organization. Volcani Center. Bet Dagan. Israel'

.

I Introduction .................................................. I1. Potential Advantages of Trickle Irrigation ............................ A. Improving Soil-Water Regime for Greater Crop Yield . . . . . . . . . . . . . . . . . B . Minimizing the Salinity Hazard to Plants .......................... C. Partial Wetting of the Soil Volume ............................... D . Maintaining Dry Foliage ....................................... E. Flexibility in Fertilization ..................................... F . Possible Water Saving ......................................... G . Technical-Economical Features . . . . . . . . . . . . ..................... 111 Problems in Practical Use ......................................... A. Clogging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Salinity Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. System Design Problems ....................................... D . Technical Improvements ....................................... IV. Modeling of Water and Salt Flows .................................. A. The Physical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Governing Equations ......................................... C. Water Flow Boundary Conditions ................................ D . Two-Dimensional Approximations ............................... E . Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Comparison with Experimental Data . . . . . . . . ..................... V . Soil-Water Regime during Trickle Infiltration .......................... ............ A . Size of the Saturated Water Entry Zone . . . . . . . . . B . Water-Content Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Wetting Fronts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Effect of Surface Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Solute Distribution during Infiltration ............................... VII . Application of Infiltration Models to the Design of Trickle Irrigation Systems A . Transient State Infiltration ..................................... B . Steady Infiltration . . . . . . . . . . . . . . .......................... C. Estimation of Spacing between Emitters . . . . . . . . . . . . . . . . D . Numerical Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Effect of Soil Hydraulic Properties on Spacing-Discharge Relationships . . . F . Designing the Lateral System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Water Management in Marginal Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ListofSymbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ....................................................

.

344 345 346 348 348 349 350 350 350 351 351 352 352 353 353 354 355 356 357 360 363 367 367 371 371 372 372 376 377 377 378 382 382 385 387 389 391

' This review was in part prepared during sabbatical leave at the Department of Agronomy. Cornell University. Ithaca. New York . 34 3

344

ESHEL BRESLER I.

Introduction

The use of trickling or dripping as a method of irrigating large fields has become quite common practice in agricultural production all over the world. The method is now one of the fastest growing new technologies in agriculture (Gustafson et al., 1974; Halevy et al., 1973). The acreage under trickle irrigation in the United States and throughout the whole world is steadily increasing. The area under drip irrigation in the United States was 100 acres in 1970 and over 70,000 acres in 1974. The anticipated 5-year projection is for more than 217,000 acres in the United States and for at least 100,000 acres in other countries (see Table I). Trickle irrigation is being adapted to almost all types of crop production (Boaz, 1973; Dan, 1974; Goldberg and Shmueli, 1970; Gornat et aZ., 1973; Goldberg er ab, 1971; Halevy etal., 1973; Hiler and Howell, 1973; Yagev and Choresh, 1974; Waterfield, I973), to all types of land (sand dunes t o clay soils, level land to steep hilside), and to relatively saline water. New land which previously could not be used successfully for agriculture has been made available through trickle irrigation (Boaz, 1973; Goldberg and Shmueli, 1970; Gornat et aZ., 1973; Halevy et aL, 1973; Willens and Willens, 1974). A trickle irrigation system consists of emitters which distribute the water for irrigation. These are usually attached to laterals to which water is delivered through the submains or main lines (Heller and Bresler, 1973; Rolland, 1973). The trickle emitter is essentially a water energy reducer. It acts generally as a resistor which dissipates the energy of water flowing through it and thus reduces the flow rate to a given discharge. The energy loss is controlled by means of an orifice, micro-holes, “button” nozzles, slits, or pores, and by the Iength of the TABLE I Drip Irrigation Survey‘ Country

Acreage in 1974

Australia British Columbia, Canada Central America Cyprus Israel Mexico New Zealand South Africa Other countries United States

25,000 500 700 400 15,000 16,000 2,000 8,600 3,000 72,000

>217,000

143,000

>3 10,000

Total ‘After Gustafson er al. (1974).

5-Year estimate 60,000 -

3,000 -

-

30,000

TRICKLE-DRIP IRRIGATION

34 5

water-flow path. The length of the water-flow path is adjusted with a spiral or screw thread plastic tube, by “spaghetti” or maze emitters, or by means of a porous material. Vortex drippers in which water is forced tangentially into a circular chamber have been fairly recently designed (Halevy et al., 1973). The flow rate is controlled by the pressure distribution which in turn is determined by size and type of dripper and the water carrier (Karmeli, 1971; Halevy et al., 1973). The surface approach, as opposed to underground application of trickling (subsurface irrigation), was initiated in Israel. It has been successfully and widely used for the past few years (Boaz, 1973; Heller and Bresler, 1973; Gornat etal., 1973). In the surface approach the irrigation emitter is placed directly on the soil surface so that the water infiltration takes place in an area which is small compared with the total soil surface of the irrigated field. As a result one has a case of three-dimensional irrigation. This differs from the usual one-dimensional flooded or sprinkler irrigation where the area across which irrigation water enters the soil is considered to be identical to the total area of the irrigated field. Optimal irrigation criteria are achieved by adjusting the trickling system to the soil hydraulic properties and to the water and nutritional requirements of the specific crop grown. This is done by selecting the best known combination of all constituents which comprise the trickle irrigation system. Recent work on the mechanisms underlying the general principles of a trickle irrigation system is presented here. The basic concepts of transport phenomena in soils are related to a quantitative evaluation of the water and salt regime in trickle-irrigated soils. Emphasis is placed on applying these principles by agronomists, soil specialists, and engineers to the design of trickle-drip irrigation systems for soils with different hydraulic characteristics. A general summary of the present knowledge of the subject is provided rather than a detailed review, interpretation, and evaulation of each individual publication that has appeared in the scientific literature.

I I. Potential Advantages of Trickle Irrigation

The traditional methods of water application to the soil have advanced from simple flood irrigation, through furrow irrigation, to a scientifically controlled sprinkler irrigation system. Each of the conventional irrigation methods has certain advantages and limitations when one considers their technical, economic, and crop production values. The trickle irrigation method has been developed for specific conditions o f an intensive irrigated agriculture. Some of the technical and agronomic advantages that may be achieved by selecting the trickling method are described in the following sections.

346

ESHEL BRESLER

A. IMPROVING SOIL-WATER REGIME FOR GREATER CROP YIELD

The traditional irrigation cycle under furrow, flood, or portable sprinkler system consists of a relatively short period of infiltration followed by a long period of simultaneous redistribution, evaporation, and extraction of water by the growing plants. In t h s mode of irrigation there is a fixed cost associated with each water application and therefore one’s goal is to minimize the number of irrigations by increasing the time interval between two successive irrigations without causing an economic yield reduction. To minimize irrigation frequency the usual goal was .to maximize the quantity of available water stored in the soil for subsequent water use by the crop before the next irrigation. The economic constraint of the traditional irrigation methods, which cause extremely large time fluctuation in the soil-water potential (Bresler and Yaron, 1972), has been partly removed by the solid-set and central pivot irrigation systems and by the development of trickle irrigation systems capable of delivering water to the soil in small quantities as often as desired with no additional cost (Rawlins, 1973). As the frequency of irrigation increases, the time-average soil-water potential increases and is restricted to a narrow range, hence eliminating low average soil-water content and high water fluctuations as factors affecting plant growth and crop yields. This increase in soil-water potential (or decrease in the total soil-water suction) at very frequent or continuous water application is a consequence of both high average matric potential and low soil solution concentration resulting from the fact that the salinity of the soil solution approaches that of the irrigation water. Ayers e l al. (1943), Wadleigh and Ayers (1945), and Wadleigh et al. (1951) suggested that matric and osmotic potentials are additive in their effect on plant growth. There is some evidence to support the view that crop yield of many crops is increased by maintaining the soil-water regime at high time-average values of soil-water potential in the effective root zone (Rawitz, 1970; Hillel, 1972; Childs and Hanks, 1975). The general increase in the total soil-water potential with irrigation frequency suggests that crop yield may be highly increased by very high irrigation frequency. The maintenance of continuously high water potential, thus minimizing fluctuations in the soil-water contents during the irrigation cycle, may be an important and advantageous feature of trickle irrigation if the yield response curve to water is convex and the effect of fluctuation in the water status on the crop yield behaves similarly to the theory of Zaslavsky and Mokady (1966) and Zaslavsky (1972), as follows. It is a well-established assumption (e.g., Taylor, 1952) that crop yield-irrigation relationships depend on both the average of some index of the soil-water regime 5 and the time deviations from 6.Generally, this can be expressed as

Y(@)=

ml

(1)

34 7

TRICKLE-DRIP IRRIGATION

where Y is crop yield, Qi is the soil-water regime index (e.g., sum of matric and osmotic potentials), & is its time average (assuming uniform 4, with respect to space), t is time and @ is the time (daily) deviation of @(t)from &, i.e., @(t) = @(t)- 6

(2)

Note that when there are no fluctuations in Q, (i.e., q5 = 0), then @(t)= 6 and the yield response function t o water becomes

Y(@)= Y(@= 6,O)= Yo(&)

(3)

where Q, is perfectly uniform at the 6 level. Expanding Y(@)function about Qi = & by Taylor’s series and neglecting third-order and higher terms, we obtain [@Wl az Y ( 6 ) ay Y ( 6 + #) = Yo($) + #(t)- (&) t - (4) a@ 2 aQZ Averaging the yield over time

[

,=l T

i

-

Ydt=Yo(@)+-

t=0

T

@(t)dt+

0

2T

1

[@(t)]dt (5)

0

The first term on the right-hand side (RHS) of Eq. (5) is identical to Eq. (3), i.e., yield response function without fluctuation in @; the second term on the RHS of Eq. (5) vanishes upon averaging because average deviations are zero. Since the integral T

T

0

is the mathematical definition of the variance u2 therefore

r

a2y F = YO(*)t-u2 2 aQ2

Here again Y is the yield, is the average yield, @ is the water regime index (e.g., sum of matric and osmotic potentials), & is its average over time, and uz is the variance of @. Note that the first term on the RHS of Eq. (6) is the yield that would have been obtained with perfectly uniform levels of @ all at the average 6 (yield without fluctuations), and the second term is the first correction to the yield due to fluctuations (fluctuation contributions). Note also that if the Y ( @ )function is convex, then the second derivative in Eq. ( 6 ) is negative and time fluctuations in the soil-water potential will cause a crop yield reduction. Generally, a’ Y / M 2 < 0, and thus fluctuations of soil-water content or of total water potential with time have a negative effect on the crop yield. If this is indeed the general case, then the best irrigation policy is to apply the water as frequently as possible (Rawlins and Raats, 1975) as long as there are no aeration

348

ESHEL BRESLER

problems (Dasberg and Steinhardt, 1974). Referring to Eq. ( 6 ) it is clear that the crop yield increases with increasing irrigation frequency if the yield response to soil-water potential is a convex monotonic increasing function. With the trickle irrigation method, in addition to solid-set and central pivot irrigation systems capable of delivering water t o the soil as often as desirable with no additional costs (Rawlins and Raats, 1975; Heller and Bresler, 1973), this potentiality in increasing crop yield can be achieved. It is possible, therefore, to draw a conclusion that if the views expressed by Zaslavshy (1972) and given in Eq. ( 6 ) will consistently be supported by experimental evidence (such as that of Patterson and Wierenga, 1974, in the experiments performed in 1972 but not in 1973), then optimizing the soil-water regime for greater crop yield may be an important advantage of the trickle-drip irrigation method.

B. MINIMIZING THE SALINITY HAZARD TO PLANTS

Recent work by Bernstein and Francois (1973) showed that brackish irrigation water (2450 mg/liter total salts) can be used successfully in drip irrigation to obtain almost the same yield as nonsaline good quality water. Using the same water for furrow and sprinkler irrigations caused yield reductions of 54 and 94%, respectively. Increasing the irrigation frequency caused only an 18 and 59% reduction in yield for furrow and sprinkler irrigation, respectively. Minimizing the salinity hazard to plants irrigated by trickling can be related to: (a) the displacement of salts beyond the main efficient root zone (Patterson and Wierenga, 1974, Fig. 10; Tscheschke et al., 1974; Yaron et al., 1973); (b) lowering the salt concentration by maintaining a relatively high soil water content due to the high frequency irrigation; and (c) avoiding leaf “burning” and damage due to salt accumulation on the surface of leaves which are in contact with irrigation water. Bernstein and Francois (1975) attributed higher yield loss and injury of bell peppers (Capsicum fmtescens) irrigated by sprinkling as compared with trickling primarily to foliar salt adsorption rather than to osmotic shock caused by flushing the salt-which had accumulated at the soil surface between two successive irrigations-into the root zone.

C. PARTIAL WETTING OF THE SOIL VOLUME

An additional feature of trickle irrigation is the possibility of restricting water supply to those parts of the soil where the activity of the root system, with respect to water and nutrients, is the most efficient (Dasberg and Steinhardt, 1974). Bernstein and Francois (1973) found that many of the roots under trickle irrigation occur in the surface 2.5 cm of soil, except when salt accumula-

TRICKLE-DRIP IRRIGATION

349

tion inhibits root development. Black and West (1974) tested the effect on water use of young apple trees when varying proportions of the root systems were supplied with an “optimum” water regime. They found that when the fraction of the wetted root system was decreased from 1.0 to 0.25, the relative transpiration reduced from 1.0 to only 0.75. Their results also suggested that wetting substantially less than the total root system daily would produce at least as good a regime for plant water supply as would wetting the entire root system with a 14-day interval between irrigations. Glasshouse and growth-cabinet experiments (Frith and Nichols, 1974) showed that local application of water to less than the total root volume did not affect the ability of the tree to take up sufficient water, and that roots in the wetted root zone increased their ability to take up water. These results are similar to those of Lunin and Gallatin (1965). It was also found by Frith and Nichols (1974) that a portion of the root system could, if required, assimilate as much nitrate nitrogen as the whole root system. This experimental finding is of practical importance since satisfactory nutrition can be obtained by dissolving fertilizers in the trickle irrigation water and the roots in the wetted root zone should increase their efficiency of nutrient uptake in a manner similar t o their increasing efficiency in the water uptake. Thus, under trickle-drip irrigation it is possible for trees, under certain climatic conditions, to have considerably less than the total root system wetted. The ability of a peach tree quickly and profoundly t o adapt its root system t o the partial wetting pattern of trickle irrigation is also of interest. A whole new root system for large trees (5.8 meters wide and 5 meters high) was developed in a few months and the tree continued to produce heavy yields (WiUoughby and Cockroft, 1974). Selective wetting of the soil surface has additional benefits, such as reducing water evaporation by preventing evaporation of water from outside of the wetted surface zone. The partial wetting also restricts the growth of weeds to the wetted region and thus reduces the cost of weed control by decreasing the need for weed control beyond the wetted region. Weeds at the wet spots may be controlled very efficiently by applying herbicides through the drip system. More convenient pest control is achieved by leaving dry strips on which the pest control machinery can be moved.

D. MAINTAINING DRY FOLIAGE

Dry foliage retards the development of leaf diseases that require humidity. It obviates the necessity for removing plant-protecting chemicals from the leaves by washing. This is, of course, in addition to the prevention of leaf burns due to the lack of direct contact of the leaves with saline irrigation water (Bernstein and Francois, 1975).

350

ESHEL BRESLER

E. FLEXIBILITY IN FERTILIZATION

Trickle irrigation offers flexibility in fertilization, a benefit unique to this system (Lindsey and New, 1974; Isob, 1974). Since fertilizers can easily be applied along with irrigation water, frequent or continuous application of nutrients at low concentrations is feasible and seems to be very good practice (Safran and Parnas, 1975). Optimizing the nutritional balance of the root zone is possible by supplying the nutrients directly to the most efficient part of the root zone. Other features include good fertilizer distribution with minimum leaching beyond the root zone and more options in the timing of fertilizer application than with any other distribution system. However, the fertilizer mixtures must be completely soluble in water, not leave any residues in the dispenser, and must not cause clogging of the emitters (Grobbelaar and Lourens, 1974).

F. POSSIBLE WATER SAVING

There are several ways by which water may be saved in using trickle irrigation as compared with other traditional irrigation methods (Patterson and Wierenga, 1974). Under trickle irrigation loss of water due to runoff in low permeable or crusted soils (Kemper and Noonan, 1970) is reduced. In addition, destruction of the surface-soil structure and the development of surface crust (Lemos and Lutz, 1957) is avoided and water infiltration into the soil is largely improved (Rose, 1961). Much water saving may be achieved by restricting the water supply to the extent of the most efficient root zone (Dasberg and Steinhardt, 1974). By not wetting the entire inter-row or inter-tree space, especially in young crops or trees (Dan, 1974), direct evaporation from the soil surface and water uptake by weeds are drastically reduced (Lemon, 1956). On steep hills and/or under strong wind conditions, furrow and sprinkle irrigation methods are very inefficient with respect to water saving (Seginer, 1967). Under these conditions, the use of trickle irrigation prevents water loss beyond the border of the irrigated field by wind convection (Seginer, 1969) or runoff in contour cultivation on steep hills.

G. TECHNICAL-ECONOMICAL FEATURES

In many countries and instances where labor and water have become limiting and too expensive, the method of trickle irrigation has been developed. Automation is one of the very reliable tools which can be easily used in trickle irrigation for accurate soil-water control, the supply of water as needed, and a large reduction in manpower. A simple automation system includes an automatic

TRICKLE-DRIP IRRIGATION

35 1

metering valve or an automatic timer that can be set for daily operation at certain hours of the day. More sophisticated and complicated units can be controlled by electrified tensiometers in the root zone, by having an electrical impulse trigger the system when the matric potential is reduced to a certain dryness. These days, with the energy supply becoming more limited and expensive, an optimal irrigation method should also rely on a relatively low operational pressure. This may be an important technical feature of the trickling method as long as the energy losses in the “Control System Unit” (see Heller and Bresler, 1973, Fig. 5) are not too large. An additional important technical-economical feature of trickle irrigation is the use of a small pipe diameter and the possibility of operating the system 24 hours a day, including during windy hours (Heller and Bresler, 1973). Frequent irrigation during the windy hours protects sensitive and high investment crops from cfesiccation without wasting water by wind convection (Seginer, 1969). Another economical-technical feature of the trickle-irrigation system is the reduced cost of weed control. This is so because weeds grow only in the wetted spots and weed control may be achieved through the trickling system. The use of herbicides through the irrigation system offers an answer to weed problems under trickle irrigation, especially if one uses herbicides capable of killing weeds as they germinate (Lange et al., 1974). Another aspect of trickle irrigation in relation to the economy of pest control is the possibility of using the soil fungicides in the irrigation system to control root rot fungus (Zentmyer et al., 1974).

Ill. Problems in Practical Use

The technical and agronomic advantages utilized in selecting the trickle-drip method have been discussed. However, some problems in the practical implementation of the method do still exist: some of the problems will be discussed here.

A. CLOGGING

Operational difficu ies with the trickling metho- sometimes arise from clogging of the drippers, which is the most severe maintenance problem. Clogging affects nonuniform water distribution and requires frequent replacement of emitters, which is quite an expensive procedure. Clogging is caused by several factors (Peleg et al., 1974), such as: (a) root penetration; (b) blockage of orifice by sand, rust, leaves, small soil animals, microorganisms, etc.; and (c) precipita-

352

ESHEL BRESLER

tion of soluble salts such as carbonates, iron, aluminum, and phosphate compounds. Smaller particles can pass through the filtering system and act as crystallization nuclei in the trickling system. Overcoming the clogging-caused difficulties involves the use of a very efficient filtering system and periodic cleaning or replacement of the drippers (Boaz, 1973), reverse flushing (Rawlins, 1974), and chemical treatments (McElhoe and Hilton, 1974). All these, of course, involve extra expense and manpower, especially when a large number of emitters and laterals per unit area are needed.

B. SALINITY PROBLEMS

In arid regions where saline water must be used, there is a tendency for salt to accumulate close to the margins of the wetted zone (Tscheschke et al., 1974; Patterson and Wierenga, 1974; Yaron et al., 1973). The salt which tends t o accumulate at the periphery of the wetted soil volume, midway between emitters (Gerard, 1974), may be washed by rain into the main effective root zone and may cause osmotic shock to plants (Bernstein and Francois, 1973). The accumulation of salt at the periphery between emitters could be a serious problem also for seasonal crops, because some of the newly sown plants may be found in regions of high salt concentration from the previous crop. This salt must therefore be leached before the next crop is planted (Patterson and Wierenga, 1974). In areas where there is not sufficient rainfall for the leaching process, then leaching must be accomplished by sprinkler or flood irrigation, which increases the cost considerably. Another expensive solution is to place the emitters at a much closer spacing in order to approach one-dimensional vertical flow. With high frequency irrigation it is also possible to apply water in excess of evapotranspiration in order to leach the accumulated salts out of the root zone and thus control soil salinity. Controlling the quantity of water passing through the root zone to avoid salinity buildup is one of the problems of trickle irrigation.

C. SYSTEM DESIGN PilOBLEMS

In the sprinkle irrigation method, when one-dimensional water flow takes place, there are no severe design problems. The spacing between sprinklers and the operational pressure are designed so as to meet certain uniformity criteria (Christiansen, 1942; Hart, 1961). Irrigation scheduling is designed by taking into account soil-water or plant-water criteria, one-dimensional evapotranspiration, and effective rooting depth (Hake and Hagen, 1967). In trickle irrigation, when

TRICKLE-DRIP IRRIGATION

353

three-dimensional flow occurs, the main design problem is to select the proper combination of emitter spacing and discharge for a given soil and crop (see Section VTI). In cases where there are no salinity problems, the wetted volume of soil should be kept within the borders of the efficient root zone. In order to prevent losses of water beyond the efficient root zone, one must determine the proper combination of emitter spacing and discharge, irrigation frequency, and amount of water applied for a given set of soil-water characteristics, crop root distribution, water distribution prior to each irrigation, as well as the pattern of water uptake by roots. Determination of the proper emitter spacing and discharge under given external conditions requires knowledge of soil hydraulic properties together with crop response t o size and form of the wetted soil volume and to the water distribution and fluctuation within this volume.

D. TECHNICAL IMPROVEMENTS

Technical improvements include the following aspects: (1) mechanical improvements of emitters, various fittings, and filters; (2) finding chemical and other treatments to eliminate mechanical and chemical clogging of emitters and failure of the operation system; and (3) development of a good fertilization system. In addition, development of a portable trickle irrigation system and of cheaper components (Rawlins et al., 1974; Wilke, 1974) would make the method more suitable for irrigating close-planted crops, which are sensitive to the water distribution in the soil.

I V . Modeling of Water and Salt Flows

Mathematical models are used to approximate physical conditions through mathematical equations. When the equations are too complicated to be solved analytically, their solutions may be obtained with the aid of a computer. Several phases are involved in the development of the model. These are: (a) identification of the relevant physical problems and the mathematical model that can simulate the physical problem; the physical parameters that are included in the model are determined separately and independently; (b) mathematical formulation of the governing equations and the boundary conditions of the problem; (c) development of a method of solution in order t o solve the mathematical problem; (d)verification of the model, first by a comparison between exact analytical solutions and the approximate model solution, and second by performing physical experiments and comparing the theoretical results with field or

354

ESHEL BRESLER

laboratory observations; (e) efficient implementation of the procedure on the computer if computer methods are used; (f) compatibility of the final product with existing models, if there are such models.

A. THE PHYSICAL SYSTEM

The irrigation system to be considered first consists of an emitter which distributes the water for irrigation. Water enters the emitter-which reduces its pressure-and discharges out as a trickle at a predetermined rate. The irrigation trickle emitter is placed directly on the soil surface, so that the area across which infiltration takes place is very small compared with the total soil surface. As a result, one has a case of three-dimensional infiltration of water into the soil. This differs from the usual one-dimensional case of flood or sprinkler infiltration, where the area across which water enters the soil is assumed to be identical to the entire soil surface. As mentioned earlier, one of the potential advantages of trickle irrigation is to maximize the time-average soil water potential by increasing irrigation frequency. As the frequency increases, the infiltration period becomes more important and the irrigation cycle is changed from an extraction-dominated process to an infiltration-dominated process (Rawlins, 1973). Since, in trickle irrigation, when irrigation is sufficiently frequent, the irrigation cycle is dominated by the infiltration stage, the discussion here is limited to modeling of salt and water flows during infiltration only. Consider a field (Brandt et aZ., 1971) that is irrigated by a set of emitters or trickle sources, spaced at regular intervals, 2X and 2 Y , as shown in Fig. 1. Due to the symmetry of the pattern, one can subdivide the entire field into identical volume elements, W, of length X , width Y, and depth 2, where the latter always remains below the wetting front. Here, each volume element acts as an independent unit in the sense that there is no flow from one element to another.

FIG. 1. Schematic representation of a trickle-irrigated field.

TRICKLE-DRIP IRRIGATION

355

Therefore, in order to describe the salt and water flows in the entire field, it is sufficient to analyze their status in a single element, W. This, of course, is true only for the interior part of the field that is not too close to the margins.

B. GOVERNING EQUATIONS

The differential equations governing the flow of water and noninteracting solutes in an unsaturated soil system can be written in indicial notation as (cf. Neuman, 1973; Bear, 1972):

Here xi (i = 1,2,3) are spatial coordinates (xj the vertical considered to be positive downward), 0 < Kr < 1 is the relative hydraulic conductivity (Neuman, 1973), K:j is the hydraulic conductivity tensor at saturation, 0 is volumetric soil-water content, t is time, p is pore water pressure head, Dij is coefficient of hydrodynamic dispersion (combining the effects of diffusion and mechanical dispersion), q is specific flux of the soil solution, and c is solute concentration in the solution. Equations (7) and (8) are written in an indicial notation such that quantities with a single subscript, or index, represent components of vectors; quantities with two subscripts are components of tensors; and when an index appears twice in any given term this term must be summed over all admissible values of that particular index [such as i and j in Eqs. (7) and (8)] . The form of the dispersion term Dij in Eq. (8) has been the subject of intense discussion. Recent experimental and theoretical studies (Ogata, 1970; Perkins and Johnston, 1963; Bear, 1972) suggest that in isotropic and homogeneous porous media the principal axes of dispersion are oriented parallel and perpendicular to the mean direction of flow. T h s indicates that for such media the transport of the dispersed material can be defined by two characteristic dispersion components that are specified when the mean direction of flow is known. Thus the hydrodynamic dispersion coefficient Djj for isotropic media can be defined similarly to the definition by Bear (1972, p. 612) as

oij= hT1 v 1 6t~(A, ~ - hT) vpy~vi t D p ( e )

(9)

where X, is the longitudinal dispersivity of the medium, AT is the transversal dispersivity of the medium, 6 i j is Kronecker delta (i.e., 6ii = 1 if i = j and 6ij = 0 if i # j), & is the d-th component of the average interstitial solution velocity Y,

356

ESHEL BRESLER

and D p ( e ) is the soil diffusion coefficient as defined by Bresler (1973) using Eq. (57) of Olsen and Kemper (1968).

C. WATER FLOW BOUNDARY CONDITIONS

Referring to Fig. 1, we shall place the origin (O,O,O) of the coordinates at the center of a particular emitter (trickle source) and define W as the domain W = 0 < x = x l < X , 0 < y = x2 < Y , 0 < z = x g 1 . I t is clear t h a t x = O , x = X , < y = 0, and y = Y are planes of symmetry, for which the normal derivative of 0 must vanish, and where no flow exists across these boundaries. If one also assumes that below the wetting front (at the depth z = Z), M / a z = 0 is a good approximation for the period of the infiltration, or at least that imposing this condition would have a negligible effect on the region of interest (Brandt et al., 1971), one has the following no-flow boundary conditions formulated for Nx,y,z,t) as

I.

{

aO/ax= 0 a t x = 0 and x = X for t > O

a e / a y = O a t y = 0 and y = Y f o r t > O ae/& = 0 at z = z for t > 0

(10)

In order to define the boundary conditions at the soil surface (z = 0), the discharge from the trickle source must be known as a function of time. This rate of discharge is denoted by Q(t) and will be referred to as trickle discharge. In addition, an assumption must be made concerning the horizontal area across which water infdtration takes place. It has been observed (Bresler et al., 1971) that in general a radial area of ponded water develops in the vicinity of the trickle source. This area is initially very small, but its radius ( p ) becomes larger as time increases. Since the ponded body of water is usually very thin, one can safely neglect the effect of storage of water at the soil surface. This means that the water from the trickle source is able to infiltrate into the soil, or evaporate into the air, instantaneously. Obviously, the soil-water content immediately beneath the ponded area is always equal to the water content at saturation, €Is. This saturated area is the only place where water can.infiltrate into the soil element, W.Thus it will be referred to as the saturated area, or the zone of water entry. It: is assumed that the center of this disklike zone is at (O,O,O) (Fig. 1) and that its radius p(t) is a function of time. The only additional boundary condition that must be satisfied at the soil surface outside the saturated area of water entry is that the water flux be equal to a given rate of evaporation, E. Therefore, the boundary conditions that must be satisfied at the soil surface are, “moving boundary conditions,” and they can be mathematically formulated for all t > 0 and at z = 0 as

TRICKLE-DRIP IRRIGATION

e = e, q e ) + E -K(s)&= aZ o

for

o < x 2 t y z G [p(t)] x2 + y 2 > [p(t)l2

for aP 1 /[K(B,) + E - K(0,) az 1 dx dy = z Q ( t >

357 (1 1) (12) (13)

G

Here, G is the quarter disk defined by x z + y 2 S [p(t)]2 , x,y > 0 ;0, is the water content of saturated soil; p(t) is the radius of the ponded or water saturated area as a function of time; E = E(t), is a given evaporation function; and Q(t)is the time-changing discharge of the trickle source. The large-scale field flow problem is now confined to the cube

w =( o < x s x , o ~ y < Y , o < z < z ) , on the sides of which it is possible to formulate suitable boundary conditions. To these we have to add the initial conditions 8 = 8,

in W, for t = O

(14)

where 0, is the initial soil-water content. D. TWO-DIMENSIONAL APPROXIMATIONS

At present, the problem defined in Eqs. (7) and (10) through (14) can be solved only by numerical methods with the aid of a high-speed digital computer. Excessive amounts of computer time are needed to solve the four-dimensional grid (x,y,z,t) in this kind of problem. The expense of long computer runs can be greatly mitigated by considering special cases of the three-space dimensional problem which are amenable to two-space dimensional modeling. Also two-dimensional experimental data for comparison with simulated results are more easily obtained. Such a two-dimensional flow is the cylindrical flow which takes place in the field as long as the wetting front has not reached the outer boundaries of the volume element W. Other cases of a two-dimensional problem are: (a) a line of trickle sources very close t o one another, and (b) a line of perforated or porous tube. Here, the problem can be viewed essentially as one of a plane flow. Thus, two mathematical models will be considered in this review: (1) a “plane flow” model involving the Cartesian coordinates x and z, and (2) a “cylinder flow” model described by the cylindrical coordinates I and z. 1. Axisymmetric Cylindrical Flow Models

In the axisymmetric cylindrical flow model the case of a single trickle emitter (or a number of emitters spaced at sufficient distance apart) is considered. The

358

ESHEL BRESLER

origin is at the center of the epitter and c and 6’ are functions of the coordinates x = x3, t, and r = (x2 t y2)”’ = (x: t x $ ) ” ~ The . axial symmetry conditions and assuming the soil to be a stable, isotropic, and homogeneous porous medium, with Darcy’s law applied in both saturated and unsaturated zones, cause Eqs. (7) and (8) to take the form:

Here, Y is the radial coordinate, z is the vertical coordinate (considered to be positive downward), K(0) = Kr(B)KS is the hydraulic conductivity of the soil (depending on 0 alone), and H is the hydraulic head (sum of pressure head, p , and gravitation head, z ) . Note that Eq. (15) considers the water pressure head p and the hydraulic conductivity K to be single-valued, continuous functions of 0 and the hydraulic gradient to be the only force causing water to flow. Also note that in the development of Eq. (16) one usually assumes that solute transport is governed by convection and diffusion, and that DIP,D,, = D,, and D,, are given by Eq. (9) with r and z substituted for i and j , respectively. The initial and boundary conditions for e(x,r,f) [Eq. (15)] in the cylindrical element W = (0 < r
ae

-=O; az

8=8,; E-K(O,)--O; aHaz 27r

O
O
t=O

r = O , r = R ; O G z < Z ; O
(174 (17b)

O
z=Z;

0
(17c)

O
z=O

0
(1 7d)

p(t)
0
(17e)

[E -K(B,)$

aH

rdr = Q(t)

0

where T is the end time of infiltration. The boundary and initial conditions for c(r,z,t), [Eq. (16)] under the axisymmetric flow conditions of infiltration from trickle sources (Bresler, 1975), are

Finally, the initial conditions are c(r,z,O) = c,(r,z)

in

Here, q,(r,O,t) is the specific downward flux of water at the soil surface as given by Darcy’s law, C, is the solute concentration at the inlet of the trickling water, cn(r,z) is the predetermined initial soil solution concentration, and Drz(r,O,t), D,,(r,O,t) are the hydrodynamic dispersion coefficients at the soil surface, the sum of diffusion and mechanical dispersion coefficients as given by Eq. (9).

2. Plane Flow Model Consider: (a) An infinite straight line of perforated or porous tube, or (b) a set of drippers that are spaced very close to each other at equal intervals along an infinite straight line, so that their ponding areas overlap after only a short period of time. One can assume that the total ponding area has the form of an infinite strip of width p(t). Let this strip be oriented along they = x2 axis, and let x = x1 be the horizontal coordinate normal t o y then the flow becomes independent of the y coordinate. Considering the plane flow model. and the aforementioned assumptions the governing Eqs. (7) and (8) now become

360

ESHEL BRESLER

ae = a [~(e):] at

ax

t

aza [zqe)Y az

ac ac a ac ac a(ce)- a (Dxx- + D,, -- qxc) t - (LIZ,- t Dzx- - q,c) (20) at ax ax az az aZ ax where the subscripts x and z denoting the flow direction x l and x 3 , respectively and D,,, Dxz = D,,, and D,, are given in (9). Note that Eqs. (15), (16), (19), and (20) do not include sinks or sources due to uptake by plants or precipitation, dissolution, and adsorption of solute. Note also that the variable y must now be eliminated from the boundary conditions (10) through (14), and that x is substituted for r, and X for R, in (18). Of course, 2n and r in the last term on the left-hand side (LHS) of (180 must also be omitted. Notice also that Q(t) is now the trickle discharge per unit length of the strip (cm3 cm-’ rnin-’ ). This plane flow model is relatively easy to reproduce in the laboratory and can therefore serve as a convenient tool for comparison purposes. Such a comparison between laboratory and theoretical results will be described in Section IV, F of this chapter. E. SOLUTIONS

1. Numerical Approaches The problems defined by the nonlinear partial differential Eqs. (15) and (16) or (19) and (20) together with the associated boundary conditions given by Eqs. (17) and (18) can, at present, be solved only by numerical methods with the aid of a computer. Brandt et al. (1971) used an approach that combined the noniterative alternating-direction implicit (ADI) difference method with the iterative Newton method to solve numerically for values of @(y,z,t), q,(y,z,t), and 4,(y,z,t), where y is introduced for x when dealing with plane flow and for r when the flow is cylindrical. The calculated values can then be used to obtain c(y,z,t) from Eq. (16). To solve for c(y,z,t) by the second-order finite difference approximation to Eq. (16) or (20) and Eq. (18), the values of D,,(y,z,t), DYz(y,z,t), and D,(y,z,t) must be known (Bresler, 1975). After B(y,z,t) and q(y,z,t) are known, one may calculate V7(y,z,t) = q7(y,Z,t)/e(y,z,t)as well as V,(y,z,t) = q,(y,z,t)/O(y,z,t) and V = + and so estimatesD,,, D,,, and D,, from Eq. (9). The methods used to solve for O(y,z,t) (Brandt et al., 1971) and c(y;z,t) (Bresler, 1975) resulted in a numerical technique that is simple, efficient, unconditioned stable, and second-order accurate.

(c G)l’’;

2. Linearized Water Flow Solutions L

Solutions for transient and steady infiltration from point, line, strip, and disk sources which can be applied to simulate trickle irrigation cases have recently

TRICKLE-DRIP IRRIGATION

36 1

been published (Wooding, 1968; Philip, 1971; Raats, 1971, 1972; Warrick, 1974; Warrick and Lomen, 1974, 1976; Lomen and Warrick, 1974). The linearization of the nonlinear differential Eq. (15) or (19) is attained by applying the transformed water content S(0) function similar to the matric flux potential SO?) (Gardner, 1958) as S@)=?

Po

KO?)dP

(21)

when po is a reference pressure defined by p o = p ( 0 , ) . Usually the reference value is chosen such that po + - w. In practice when water content varies over a limited range it is sometimes possible to approximate the nonhysteritic hydraulic conductivity function , KO?),as K07) = K , exp (ap)

(22)

where K , is usually the saturated hydraulic conductivity and a is a constant characteristic of the soil. Talsma (1963) showed that hydraulic conductivity of some field soils can be represented by Eq. (22) with values of a within the range 0.002 to 0.2 cm-’ (Philip, 1968; Braester, 1973; Bresler, 1977). Substituting Eqs. (2 1) and (22) into the nonlinear Eqs. (15) and (19) resulted in differential equations in the form

a2s -ae= - + - - a -a2s ax2 a 2

as

ax For the transient case, Eqs. (23) and (24) can be linearized by assuming that de/dS is a constant (Warrick, 1974; Lomen and Warrick, 1974). For the steady state flows the left-hand side is zero and thus Eqs. (23) and (24) reduce to linear differential equations as used by Wooding (1968) and Raats (1972) for circular and line sources, respectively. Solutions of (23) and (24) for strip, disk, point, and line sources have been obtained by Warrick and Lomen (1974, 1976), Warrick (1974), and Lomen and Warrick (1974). They assumed zero initial conditions (S = 0 at t = 0), and S was required to vanish at large distances from the sources. The boundary conditions at the soil surface (z = 0) were that of no vertical flow (except for the source) at r # 0 or x # 0, and that at r = 0 or x = 0 the surface source is allowed to change discretely with time. The latter enables one to simulate “on”-“off’ pulses in the irrigation cycle (Warrick and Lomen, 1974). The advantages of these linear solutions are the existence of analytical solutions and the possibilities of adding single solutions together to represent complex distribution of sources (Warrick and Lomen, 1974). The main limitaat

362

ESHEL BRESLER

tions are the error involved in the linearization procedure and the assumptions concerning the soil surface boundary conditions which are inadequate for the trickle-drip irrigation problem. As mentioned before (Section IV,C), the actual trickle source does not behave as an idealized point source since the water discharging from the emitter spread over a finite saturated area of the soil surface. It is more appropriate then to simulate trickle irrigation by an infiltration from a shallow circular pond. Solution to steady state infiltration from such a circular pond has been presented by Wooding (1968). He used the steady state form of Eq. (23), i.e., ae/ar = 0 with boundary conditions appropriate to steady infdtration (the time variable [t] is excluded) similar to Eqs. (17a-f), but considered a semi-infinite region with fixed saturation water entry zone (instead of prescribed discharge). Thus at the soil surface (z = 0) over a constant ponded water entry area, the soil is saturated, or in terms of the transformed variable S(P), 0

z=O;

OGrGp,;

S(O)=J K ( p ) d p = S , = -Ks - K O Po a

(254

where S, = S(p = 0) is the value of S in saturated soil, and pu is the ultimate radius of the ponded area. Beyond the water entry zone over the nonwetted area of the soil surface, the vertical water flux is zero if evaporation may be neglected. This means that in this region

aP K--K=O

as

--K=0.

or

a2

aZ

From Eq. (22) it follows that

and therefore P

l K

Po

KO

$ Kdp=-J

dK

Integrating the last equation using Eq. (2 I), yields

a During steady infiltration from a trickle source most of the soil surface in the region r 2 pu is air dry, or at least at a water content sufficiently low so that KO = K(Oo) is negligibly small, yielding K = ols. It follows, therefore, that approximately

TRICKLE-DRIP IRRIGATION

atz=O;

pu
as ---orS=O az

363 (25b)

If one also assumes that at large distances from the source, the low initial water content remains constant, one has zz

trz

+~,s=o

It is generally convenient to take the radius pu as the length unit and t o define dimensionless cylindrical coordinates .$ = r / p , and 5' = z/p,. The boundary conditions (25a-c) can then be written in terms of [ and 5' as

S(t,O = s(e,>, t2 + t2-b

O0

The problem was solved by Wooding (1968), who reduced the boundary conditions t o a system of dual integral equations and used a modification of Tranter's (1951) method. His solution is restricted to values of crp, < 10, which are sufficiently good for all purposes of practical application to the theory. For example, (Y = 0.1 the value of pu is less than 100, which allows at least 2 meters spacing between emitters. For any smaller values of cr the maximum possible spacing is obviously larger. The problem of steady infiltration from a field of equally spaced line sources was analyzed by Raats (1971). He used Eqs. (21) and (22) to derive hisgoverning equations similar to Eq. (24) with &Ofat = 0. His analytical solution showed that the flow pattern is a unique function of aix. Thus, when steady state plane flow trickle irrigation is considered, the main flow features depend both on soil characterization, a,and on the distance between the laterals or trickle lines, X.

F. COMPARISON WITH EXPERIMENTAL DATA

The solutions of the aforementioned mathematical models must be compared with experimental results in order to establish the reliability of the theoretical models, to evaluate the physical assumptions involved in them, and to ascertain the validity of the views expressed in the theories. There have been no comparisons made between analytical solution and laboratory or field results reported in the literature. Very little information has been obtained regarding a comparison between numerical solution results and experimental data (Bresler el al., 1971; Bresler and Russo, 1975). Laboratory experiments conducted under conditions similar to those assumed in the two-dimensional plane flow models (i.e., with Y < X so that Y + 0 was practically correct) have been described by Bresler ef al. (1971) and by Bresler

364

ESHEL BRESLER

a=0.983 crn’

cm-’ mi6’

L

FIG. 2. Vertical water-content distribution, e(0,z) in the plane of symmetry (x = 0). Computed results (solid lines) are compared with experimental data of Gilat loam soil (scattered points). The numbers labeling the lines indicate infiltration time (0;Q indicates the discharge per unit length. Note that the zero vertical plane (z = 0) is shifted and the data are accordingly translated along the z axis. After Bresler et ul. (1971).

365

TRICKLE-DRIP IRRIGATION

and Russo (1975). Quantitative comparisons between the theoretical and experimental water-content results are given in Fig. 2 , where the water-content profiles in the z dimensions are shown. This figure shows several sets of experimental data for each constant value of discharge rate (Q) together with the corresponding theoretical continuous curves. The theoretical results are calculated by the numerical method of Brandt et at. (1971) with the aid of soil-water parameters K ( 0 ) and p ( 0 ) of Gilat loam soil (Bresler et al., 1971). The comparison between the calculated and experimental data was made for all times and points for which there were water-content determinations, and at all the tested trickling rates. In Fig. 2 the water-content distribution along the center vertical plane [O(O,z)] is presented for five different measurement times ( t ) and at four discharge rates (Q) given as cm3 cm-' min-' due to the plane geometry. Note that the zero point ( z = 0) was shifted and the data were accordingly translated along the z axis, in order to distinguish between the different sets. The shifting of the data provides easier identification of the experimental points which corresponded to the calculated lines. The quality of the agreement between calculated and measured water-content distribution can be observed from Fig. 2 . In the case of infiltration into an air-dry soil, a clear boundary exists between the wetted zone and the dry zone in the wetting front. The wetting front is easy to determine experimentally with a minimum error. Furthermore, the wetting front is important from a practical point of view since it shows the boundaries of the irrigated soil volume. In the laboratory experiment with the initially air-dry HORIZONTAL DISTANCE X (cm)

5

10

15

20 1

L

1

20

FIG. 3. Computed wetting front position (solid lines) as compared with the observed one (dashed lines) for four infiltration times (the numbers labeling the lines). After Bresler and Russo ( 1 975).

366

ESHEL BRESLER

Gilat soil (Bresler et aL, 1971; Bresler and Russo, 1975) it was easy to identify the wetting front visually through the Perspex plates. For comparison, the location of the wetting front in the corresponding numerical solution was defined as a ridge (or line of maxima) of lgrad 0 I. Figure 3 shows a comparison, with a relatively good agreement, between the location of the calculated wetting front and the observed one as a function of the space coordinates (x,z) and the total infdtration time. The total infiltration time is indicated on each wetting front line that appears in the figure. The fact that the agreement between calculated and observed water-content distribution (Fig. 2) and wetting front (Fig. 3) is generally good suggests that model computations of water flow have little effect on discrepancies or agree-

1

-0.2

1

1

1

II

0.2

0.4 0.6 0.8 RELATIVE CONCENTRATION (C-Col /Cn

0.

FIG. 4. Vertical salt concentration distribution in terms of (c - C,)/c, in horizontal planes. Computed “plane flow” results (solid lines) are compared with measured chloride data (dashed lines). Note that the zero vertical plane (z = 0) is shifted as in Fig. 2. After Bresler and Russo (1975).

TRICKLE-DRIP IRRIGATION

367

ments between measured and computed salt flow data (Fig. 4). The comparison between measured chloride concentration and computed solute distribution data after T = 160 minutes of infiltration is given in Fig. 4 (Bresler and Russo, 1975). To be consistent with Fig. 9 the salt distribution data are expressed in a dimensionless relative form, (c - Co)/cn,where c and c, are the concentration in the soil solution at t = T and t = 0, respectively, and C, is inlet concentration. In Fig. 4 the data are presented along the vertical axis ( z ) at five different horizontal distances from the source: x = 0, 3, 7, 11, and 15 cm. Note that here also the zero point (z = 0) was shifted and the data were accordingly translated along the z axis in order to facilitate the comparisons and to distinguish between the different sets. The agreement between theoretical and measured salt distribution results is shown in Fig. 4. The agreement between laboratory and numerical water and salt flow results, which is as good as one could expect for laboratory techniques (Bresler et al., 1971; Bresler and Russo, 1975), shows that two-dimensional transport of solute and water during nonsteady trickle infiltration may be quantitatively predicted by the theoretical models. However, the prediction capability of the model and its applicability are restricted to the conditions which prevailed in the experiment and should be tested against further observations. In addition, the experimental data from a limited number of laboratory studies might be insufficient for general detailed model verification.

V.

Soil-Water Regime during Trickle Infiltration

The effect of any irrigation method on the soil-water regime of a given soil depends primarily on the conditions prevailing at the soil-surface boundary. In the case of trickle irrigation, these conditions may be defined by the trickle discharge (Q) measured as the amount of water per unit time (or amount per unit time per unit length of laterals), and by the hydraulic properties and rate of evaporation at the soil surface which determine the horizontal area across which infiltration takes place. The rate of evaporation becomes an important factor only when the potential evaporation is extremely high and the saturated hydraulic conductivity of the soil is very low. A. SIZE OF THE SATURATED WATER ENTRY ZONE

As already mentioned, it was observed that during trickle irrigation in general, a radial area of ponded water develops in the vicinity of the trickle source. This area is initially very small, but its radius becomes larger with time. Water from the emitter is able to infiltrate through this area into the soil, or evaporate from

368

ESHEL BRESLER

it into the air, instantaneously. The soil-water content immediately beneath the ponded area, A = n p 2 , is equal to the water content at saturation, OS. This saturated area, the size of which is-a function of time, is the only place where water can infdtrate into the soil from the surface. Figure 5 shows the calculated vertical water flux at the surface across the saturated water entry zone, as a function of infiltration time ( t ) and distance from the center of source. Since 0 = 0, in the interval 0 < x < p(t), the hydraulic conductivity of the saturated zone is constant and equal to the saturated conductivity. Thus, the decrease in vertical water flux with time (Fig. 5) is due only to the decrease in negative value of the vertical hydraulic gradient at the .Of -

.O i

2 cm

o=o.ge3 crn' cm-' min-'

.o 6

-.o :

. C

'i

-

.O.c

x

3

2.03

.O i

.o 1 0

I 200

400

I 600

eoo

I

I

I

I

I

I

1

1000

1200

1

1x10

.01 --K@s)

I

I

I

I

TRICKLE-DRIP IRRIGATION

369

saturated zone. Within the time considered, the hydraulic gradient has always been less than unity (the vertical flux of Fig. 5 is always greater than K,) but tends to approach a limit. The limiting value of the hydraulic gradient and the length of time involved in obtaining it depends on both the distance from the trickle source and its rate of discharge (Fig. 5). The larger the trickle discharge and the smaller the distance from the source, the faster the hydraulic gradient as a function of time approaches its limiting value. If a value of 1 to this limit were approached throughout the whole ponded area, then we would have from Eq. (170 p(t m) + [Q/K(0,)n]'/2. However, since at finite value of time, the limiting hydraulic gradient is less than 1, then p ( f ) < (Q/nK,)'12 in cylindrical flow [p(t) < Q/(2K,) in plane flow], if evaporation is not too significant. (If evaporation is important, then p(t)< {Q/[K,n + E ] "2.),Neglectingevaporation the difference between p ( t ) and [Q/(K,n)]'/2 [or Q/(2K,)] depends on the average vertical water pressure gradient at z = 0 over the interval 0 G r (or x) < p ( t ) [Eq. (29)]. Since the rate of change of this average value decreases with time (Fig. S), the rate of growth of the saturated water entry zone also decreases with time, as shown in Fig. 6. From this figure one can see that at a relatively large infiltration time ( t ) , the radius of the saturated water entry zone ( p ) is much less than [Q/K,n)] ' I 2 (see Table I1 for values of K,). Figure 6 further indicates that the radius of the saturated water entry zone will always be larger as K , decreases and Q increases. This fact has a very important practical implication (see Section VII). -+

I

-"E

0.4 0=20 liter/hour liter/hour

-

I

____-----

-

0

0

50 100 150 200 i0 INFILTRATION TIME, t (minutes)

FIG. 6. Radius of the saturated water entry zone p ( t ) as a function of infiltration time ( t ) for two soils and two discharge rates (Q). After Bresler (1977).

3 70

ESHEL BRESLER

Q.0.983 cm3 cm-l min" HORIZONTAL DISTANCE X, cm 32 LO 0 8 16 24 32 40

Q.0.495 cm3 cm-' min-'

0

8

16

24

I

48

8

-

16

-

-

FIG, 7. Water content filed as a function of cumulative infiltration ( V = 4')for two cases of discharge (Q). The numbers labeling the curves indicate water content (0). After Brandt et al. (1971).

TRICKLE-DRIP 1RRIGATlON

371

B. WATER-CONTENT DISTRIBUTION

T o illustrate the water-content distribution, the plane flow model is considered. Examples of water-content distribution for two cases of trickle discharge (Q), expressed in terms of discharge per unit length, are given in Fig. 7, which shows how the soil-water content changes with time and position during trickle infiltration. The illustration also shows the effect of trickle discharge on the water-content field. The saturated water-entry zone is shown by the particular line of saturated water content at the surface, where B s = 0.44. This zone is larger as the rate of trickle discharge increases. In the vicinity of the source (x = 0, z = 0 in Fig. 7), the moisture gradients increase when the rate of discharge decreases. These gradients can be calculated from the distances between lines of equal water content. This condition is reversed as the wetting front is approached. The overall shape of the wetted zone also depends on the trickle discharge. The vertical component of the wetted zone becomes larger and the horizontal component narrower as the rate of discharge decreases.

C. WETTING FRONTS

The wetting front is an important factor in trickle infiltration because it indicates the boundaries of the irrigated soil volume. Figure 8 shows the location of the wetting front, as a function of the space coordinates (Y,z) and the total amount of infiltrated water, for two different trickle discharges and two soils differing in their hydraulic characteristics (Table I1 and Fig. 1-3 of Bresler et al, RADIAL DISTANCE, r ( c r n )

40

Q=4 (2.20 Iiter/hour I

N

30

FIG. 8. Wetting front position as a function of infiltration time and cumulative infiltration water (in liters) indicated by the numbers labeling the lines, for two soils and two d i s charges. After Bresler (1977).

372

ESHEL BRESLER

1971). The total amount of infiltrated water (in liters) is indicated on the wetting front lines appearing in the figure. The trickle discharge, Q, in terms of volume per unit time, and the soil textures, are also indicated in the figure. The data presented in Fig. 8 demonstrate clearly that the rate of trickle discharge and the hydraulic properties of the soil have a remarkable effect on the shape of the wetted soil zone. Increasing the rate of discharge and decreasing the saturated conductivity result in an increase in the horizontal component of the wetted area and a decrease in the vertical component of the wetted soil depth. This is probably affected by the changes in the size of the water-entry saturated zone for each soil type and rate of discharge (Fig. 6). This ponded zone becomes larger as the soil becomes less permeable and as the trickle rate increases. The possibility of controlling the wetted volume of any particular soil, and the water content distribution within its boundaries, by regulating the trickle discharge according to the hydraulic properties of the soil (Figs. 7 and 8), is of practical interest in the design of field irrigation systems (see Section VII).

D. EFFECT OF SURFACE EVAPORATION

A possible effect of soil-water evaporation, which occurs simultaneously with infiltration, on the water distribution pattern can also be tested by numerical models (Brandt ef al., 1971; Bresler, 1975). The results of water distribution data, using the numerical procedure of Hanks er a/. (1969) to evaluate evaporation as a function of soil-water content E(B), as well as a high potential evaporation value of E, = 10 mm/day, remain essentially the same as if evaporation were completely neglected. It appears, therefore, that generally water evaporation which takes place during infiltration is not an important factor in infiltration from a trickle source. This is so because usually free water evaporation rate (E,) is on the order of 1 cm day-’ or less and K , of many “normal” soils is on the order of 1 cm hour-’, so that E,/K, is generally less than 0.04. The net water flux into the soil anywhere in the saturated water entry zone is given by qnet = K,(dH/dz) -E,. Since during trickle infiltration the value of dH/dz is generally less than -1, the ratio between outward flow due to evaporation and gross inward infiltration flow is, in most practical cases, less than 4%.However, under very dry conditions where E, is extremely high, and in a soil of very low permeability where K , is extremely low, the effect of evaporation during infiltration from a trickle source may be important.

VI. Solute Distribution during Infiltration

As mentioned before (Section III,B), the accumulation of salt between emitters may be a serious problem for crops irrigated by trickling. Control of soil

TRICKLE-DRIP IRRIGATION

373

salinity regime is therefore essential to the permanent operation of a trickle irrigation system. In addition, since fertilization simultaneously with irrigation seems to be a good practice, optimizing the nutritional regime in the root zone depends on the possibility of controlling the fertilizer salts in the wetted soil volume. Salt distribution in the soil under trickle irrigation will be demonstrated here by an example involving the case of low concentrated inflow solution in the irrigation water that miscibly displaces a highly concentrated solution originally present in the soil. Interactions between the solute and the soil matrix are ignored. Two soil media with widely different hydraulic properties and salt dispersion characteristics are discussed. The results presented in Figs. 9 and 10 were obtained from the cylindrical flow model when the hydraulic soil functions K(e) and p ( 8 ) were those of Gilat (loam) and Nahal Sinai (sand) soils (Bresler et ul., 1971, Figs. 1, 2, and 3). The soil diffusion coefficient was taken as D p ( 0 ) = 0.004 exp (loo), and the dispersivities were chosen to be AL = 0.2 cm, and AT = 0.01 cm [Eq. (9)] .The soils are assumed to have initially uniform water contents and solution concentration [c,(r,z) = 52.35 meqbiter and 0,(r,z) = 0.213 for Gilat; c,(r,z) = 300 meq/liter and 0,(r,z) = 0.0375 for Nahal Sinai] with 0, = 0.265 and 0, = 0.44 for Nahal Sinai and Gilat, respectively. The “water capacity” (0, - O n = 0.227), the initial volumetric salt content (cnO, = 11.25 meq/liter soil), and COO,,which is the minimum possible volumetric salt content at the soil inlet, are taken to be the same for the two soils. [Inlet salt concentration was C,(t) = 5 meq/liter for Nahal Sinai and C,(t) = 3.0 meq/liter for Gilat soil.] Two cases of constant trickle discharge, Ql = 4 liters/hour and Q , = 20 liters/hour, are considered. The general pattern of salt distribution in the two soils and for the two trickle discharges is demonstrated in Figs. 9 and 10. In Fig. 9 the salt concentration field is expressed in terms of salt concentration in the soil solution (as meq/liter bulk soil). Both figures show how the position of the dissolved salt field in the cylindrical flow pattern is influenced by the trickle discharge and the hydraulic properties of the two soils (Bresler et ul., 1971). The fact that the water capacity (0, - On), the initial volumetric salt content (c,0,), and the minimum inlet salt content (COO,) are identical in the two soils, makes it possible to compare the salt distribution data (Figs. 9 and 10) of these two soils. To be able to compare the effect of trickle discharges and to place the two different rates on an equal basis, the results are compared for an identical amount of cumulative infiltration (12 liters) and not for an equal time allowed for the irrigation. It should be noted that the values of OS, O n , c,, C,, Q and total infiltration used in the examples, are in the range of practical and actual field conditions. Figure 9 demonstrates the shape of the actual salt-concentration distribution. In both soils and with both trickle discharges, the solution concentration of the saturated water entry zone is identical to that of the infiltrated water. The concentration rises as the wetting front is approached and reaches its initial value

374

ESHEL BRESLER

RADIAL DISTANCE r (crn )

t

c

NAHAL SINAI (SAND)

FIG. 9. Computed salt-concentration field for two cases of trickle discharges (Q, and

Q , ) and two soils. The numbers labeling the curves indicate relative concentration in terms of (C - Co)/cn. The numbers in parentheses are salt concentrations (c), in meq/liter soil solution. The heavy solid lines indicate the saturated water entry radius, ( p ) and the peripheral heavy lines are the wetting fronts. After Bresler (1975).

TRICKLE-DRIP IRRIGATION

375

RADIAL DISTANCE r (cm) 0

5

10

15 20 25

30 35 0

5

10

15

20 25

30 35 40 45 50 55 1

5 10

15

20

NAHAL SINAI (SAND1

FIG. 10. Computed volumetric salt-content field (ce) for two cases of trickle discharges and two soils. The numbers labeling the curves indicate volumetric salt content (ce) in meq/liter bulk soil. Heavy solid lines indicate the same as in Fig. 9. After Bresler (1975).

near the wetting front boundary. It is clear that the shape of the overall wetted zone, which lies between the heavy lines (Figs. 9 and lo), is soil- and dischargedependent. Therefore, the salt distribution pattern should also be affected by these properties. Thus, the completely leached zone (c - C,)/c, = 0, remains at the water-saturated zone in the sandy soil but penetrates to a deeper depth in the loam soil. In addition, the leached part of the soil.in the vertical component of the wetted zone is deeper, and in the radial component it is narrower, as the soil becomes coarser (having higher hydraulic conductivities) and as the trickle discharge becomes slower.

376

ESHEL BRESLER

The picture for the volumetric salt-content field (Fig. 10) differs from that of Fig. 9 owing to the fact that the salt-content pattern is affected by the distribution of both water content (0) and salt concentration (c).Thus, although the pattern of the leached part, close to the saturated zone, is similar in Fig. 10 to that of Fig. 9, the accumulation part-close to the wetting front-is completely different. Here (Fig. 10) the salt quantities from the leached part of the soil are accumulated and reach a maximum at a certain distance from the source. The location of this maximum salt accumulation zone is largely dependent on both soil hydraulic properties and the discharge rate of the trickler. The size of this maximum salt content, however, is affected mainly by the soil properties and not by the discharge rate. The general pattern of salt distribution and its dependence on initial salinity, salinity of the irrigation inlet water, rate of trickler discharge, and the hydraulic characteristics of the soil (Figs. 9 and lo), are of practical interest for problems connected with the design of a field irrigation system, in order to control the soil salinity or fertility in the wetted root zone. Consider, for instance, an initially saline field being irrigated by a set of symmetrical emitters sufficiently far apart. Suppose that it is important to evaluate the leaching effectiveness of removing salts from a given root volume in which the main plant roots function. With an infiltration duration large enough, depending on soil and discharge rate, this leached zone may be sufficient for most of the roots to concentrate and function without disturbance (Tscheschke et al., 1974; Shdhevet and Bernstein, 1968). On the other hand, a large quantity of salt may accumulate to an appreciable level at a certain distance from the source close to the wetting fronts (Fig. 10). It is apparent (Fig. 10) that one can overcome this limitation to plant growth by changing the discharge of the trickler, its position, or both. Applications of the data of Fig. 9 to fertilization problems are possible. In this case the complete leached zone (c - C,)/C, = 0 may be interpreted as the part of the root zone in which the concentration of the nutrient in the soil solution is identical to its concentration in the irrigation water. Knowing t h s region is of practical importance from the point of view of efficient use of fertilizers under trickle irrigation.

VII.

Application of Infiltration Models to

the Design of Trickle Irrigation Systems

The problem of designing trickle irrigation systems involves the determination of trickler discharge rate, spacing between emitters, as well as diameter and length of the lateral system for any given set of soil, crop, and other field conditions.

TRICKLE-DRIP IRRIGATION

377

A. TRANSIENT STATE INFILTRATION

In the previous sections, analytical and numerical models of the more natural transient infiltration from trickle sources were discussed (Warrick and Lomen, 1974; Brandt ef ul., 1971). From these models wetting fronts and the pattern of two-dimensional distribution of a specific soil-water variable [S(O), p ( B ) , or B ] can be obtained for soils with various hydraulic properties and for different trickle discharges. The results, such as those presented in Figs. 7 and 8, can be applied in order to estimate the actual spacing between emitters for any given set of crop, soil, irrigation water quantity, and trickle discharge. Thus, for a particular crop and given field conditions, the optimal combination of discharge rate, amount of irrigation water, and emitter spacing can be obtained. For these purposes comprehensive numerical models (Brandt et ul., 1971; Bresler et al., 1971; Bresler, 1975) are preferred. However, in some specific problems when analytical solutions exist, they may also be applied to a complex field problem. An additional useful application of linearized analytical solution to transient flow problems is that they can serve as a check for more comprehensive numerical models (Warrick and Lomen, 1974).

B. STEADY INFILTRATION

The conditions of steady infiltration are not generally met in the field but may be approximated when irrigation is continuous for a relatively long time, or in intermittent irrigation when irrigation is very frequent. Water content pulses resulting from intermittent infdtration may be damped out a few centimeters from the source for many soils (Rawlins, 1973). This makes it possible to consider flow beyond this region to be essentially steady for continuous irrigation when infiltration takes place during a relatively long time, and for intermittent irrigation when irrigation frequency is of the order of a day or less. Of course, a truly steady flow can never be achieved during trickle infiltration but it represents an asymptotic case which can be used for practical purposes of designing trickle irrigation. The case of steady infiltration from a shallow circular ponded area on the horizontal surface of a semi-infinite soil, as treated by Wooding (1968), is considered to be a useful designing tool appropriate for application purposes. T h s is so because the actual trickle source does not behave as an idealized point source as assumed by other steady models, but the water is spaced over a finite circular water-saturated area on the soil surface. This radial saturated water entry zone is initially vely small but its radius p ( t ) becomes larger as time increases and approaches a constant value p u at some finite infiltration time (Fig. 6). The

378

ESHEL BRESLER

ultimate size of this zone depends primarily on the saturated hydraulic conductivity of the soil and the discharge rate of the dripper. An estimate of the relationships between the ultimate radius (p,) of the ponded-saturated water entry zone, the soil hydraulic properties K , and a, and trickle discharge Q, may be obtained from Eq. (64) of Wooding (1968) as

Q = T P ~ K+, 4K,pU/a

(27)

Equation (27) is equivalent to the boundary conditions in Eq. (17f) for the time in which the ultimate values of vertical water fluxes and hydraulic gradients are approached throughout the ponded area (Fig. 5). For this ultimate time we have from Eq. ( 1 7 0 (neglecting E), Q = n p i K , -+ nK,pi

Icl

(28)

where Ic 1 is the absolute value of the average vertical component of the pressure head gradient in the ponded zone defined by

In Eqs. (27) and (28) the first term on the right-hand side (RHS) of both equations represents the flow as a result of gravitation only, while the second term on the RHS represents the flow as a result of the pressure head gradients in the saturated water entry zone. Solving the quadratic Eqs. (27) and (28) for the positive root of p u , the ultimate size of the saturated zone is obtained as

It is clear from Eq. (30) that p u becomes larger as Q increases and K , decreases and that: (a) when Q + 0 then p u + 0; (b) when Q is very large, p u is large and the effects of a are negligibly small; and (c) when vertical pressure gradients are negligibly small, pu reaches its largest value of (Q/zKs)"'. Also p u decreases as a increases.

C. ESTIMATION OF SPACING BETWEEN EMITTERS

To estimate the actual spacing between emitters for a given set of field conditions, the hydraulic properties of the soil [K(8),p ( 8 ) or a, and K,] , and the desired value of critical water pressure p = p c midway between emitters must be known or estimated for any particular case. Using this information one can calculate wetted soil volume (Fig. 8) or the distribution of p , 0 , or S, i.e., p(r,z), 0(r,z) (Fig. 7), or S(g,a), from which the desired spacing can then be determined. For convenience and general designing

TRICKLE-DRIP IRRIGATION

379

purposes, it is sometimes sufficient to have the distribution of p , 8, or S at the soil surface, as given by 8(x,O) in Fig. 7 or by S(l,O) in Fig. 11. Figure 11 was reconstructed from Wooding (1968) and gives the calculated distribution of S(t,O)/S, for seven values of a = apu/2. Because a unique relationship between S and 8 and between 0 and p (nonhysteritic case) exists, the data in Fig. 11 represent the soil surface distribution of water content as well as the water

DIMENSIONLESS RADIUS E

FIG. 11. Soil surface values of S/S, as a function of 5 = r / p , for seven different values of labeling the lines).

a = aU/2(the numbers

380

ESHEL BRESLER

pressure head. To obtain p(r,O) and e(r,O) values from Fig. 11, when a K,, and pu are known, one has to use Fig. 11, Eq. ( 2 2 ) and the relationships

The values of 0 can then be taken from the O ( p ) relationship. It should be noted that due to the linear steady state form of Eq. (23), solutions from a single source as given in Fig. 11 can be added together to solve for a more realistic situation of multiple sources field. Furthermore, the general solution to the linearized steady state form of Eq. (23) or the flow equation (1 5) can be used for many particular and practical field problems, although it includes some errors owing to the linearization procedure and to the steady flow assumption. The assumptions of no surface water flux away from the water entry zone and of a constant (Y value may also be inadequate for some field problems (Bresler, 1977). In addition, it cannot be claimed that Eq. (22) is universally exact, but it does model the observed rapid nonlinear decrease of K with p in unsaturated soil (Philip, 1968). Values of a are generally smaller in fine-textured material and larger in coarse-textured material (Bresler, 1977), where gravity may be relatively more important than capillarity for water movement. However, in spite of these limitations the linearized steady state solution may be a useful alternative to the nonsteady numerical solution in determining the spacing between emitters in the irrigated field according to the rate of discharge, the hydraulic properties of the soil, and the desired soil water pressure between emitters. The general procedure of calculating the spacing between emitters as a function of discharge and midway critical pressuree pc involves the following steps (Bresler, 1977): (1) Estimate K ( 0 ) and p ( 0 ) or K , and a for any given soil (e.g., Figs. 2 and 3 of Bresler e l al., 1971 or Table I of Bresler, 1977). ( 2 ) Estimate p u from Eq. (30) as a function of Q for any soil using the predetermined value of K , and a,or K ( 0 ) and p(0), and calculate a = p u a / 2 . (3) Select a critical value of 0, to be used and estimate p c from the p ( 0 , ) characteristic curve (e.g., Bresler er aZ., 1971, Fig. 2) or select directly a value of pc to be used. (4) Calculate K(p,) from Eq. (22), S, from Eq. (32), and S(p,)/S, from Eq. (3 1) for any soil. (5) Obtain ,$(pc)from Fig. 11 using S(P,)/S, and a = apu/2 or from a figure similar to Fig. 7 or 8. Note that S@,)/S, = [K(p,) - K O ] / ( K , - K O ) X K(pc)/K, = exp (olpc). (6) Calculate the distance (half spacing) between emitters as a function of the

38 1

TRICKLE-DRIP IRRIGATION

I0

DISCHARGE 0 f l i t G / h o i r r )

."

FIG. 12. Estimated spacing between emitters r = rc(Pc,Q) = d / 2 for sandy soil (K,= 0.13 cm minute-', cx = 0.09), as a function of trickle discharge, Q, and selected values of pore-water pressure head (p = p,) midway between emitters (the numbers labeling the lines).

selected pressure head and trickle discharge [d = 2r,(p,,Q)] from rc = t C p u , or obtain it directly from a figure similar to Fig. 7 or 8. (7) Graph the results (e.g., Fig. 12). A figure such as Fig. 12 may be used as a practical nomogram to calculate the distance between emitters if their rate of discharge is known, to select the desired discharge when spacing is given, or to find the best spacing-discharge combination when a certain economic criterion has to be achieved. In applying the results as given in Fig. 12 only one iso-p, line must be used, since any d-Q combination depends on the chosen value of p c . This value must therefore be determined before any d-Q selection is made. The selection of p c is somewhat arbitrary and depends on the safety factor that one would like to choose. This is so because of the uncertainty involved in the response of plants to p, and to the degree of partial wetting of their root zone. It should also be remembered that in calculating the data of Fig. 12 from Wooding's method a single emitter (no interaction) was assumed. With this assumption a safety factor has already been

382

ESHEL BRESLER

taken into account regardless of the value of p c chosen. As a general guideline it is recommended that the value of p c to be selected, be the one corresponding to the transition between the lowest and highest values of a p / & (i.e., when lgrad 0 I starts to increase steeply with r). This is, of course, an approximate average p c value which may be lower (more negative) for less sensitive crops and must be higher when a specific crop sensitive to soil-water stress is grown.

D. NUMERICAL EXAMPLE

Evaluation of the applicability of any estimation method must be obtained by comparing its results with actual trickle infiltration field data. Since a good agreement exists between the numerical method and experimental field and laboratory results (Bresler et al., 1971), the steady state results of Wooding (1968) were compared with simulated transient flow data obtained by the numerical method proposed by Brandt et al.. (1971). This is, of course, a first approach which should be fully examined in the field. The parametric soil data given in Table I1 were taken from Bresler et al. (1971) for the two soils studied. In calculating K(p,) it was assumed that the relationship (22) holds for values of p which are smaller than the air entry value of pa of each soil. Values of S were calculated from Eq. (31), with K values substituted from Eq. (22) and S, values from Eq. (32), or simply by S/S, = exp(.p,). The soil parameters given in Table I1 and Figs. 1 to 3 in Bresler el al. (1971) were used to calculate the midway distance r, corresponding to p c for the two soils and two different trickle discharges using the data of Fig. 11 and the results of the numerical solution (Num. Soln.) of Brandt et al. (1971). The data presented in Table I11 show that with the described estimation methods one is able to calculate radial distances at the soil surface which correspond to a given soil-water pressure (or water content). It should be emphasized again that the proposed methods are good approximations to actual field conditions as long as the solutions simulate the actual field situations. This was previously shown in some limited, specific cases (Bresler et al., 1971).

E. EFFECT O F SOIL HYDRAULIC PROPERTIES O N SPACING-DISCHARGE RELATIONSHIPS

The effects of soil hydraulic properties on the spacing-discharge relationships are demonstrated in Figs. 6 and 13. Figure 6 , which represents the radius of the saturated-ponded water entry zone as a function of time for two discharge rates and the two soils, demonstrates mainly the effect of the saturated K , on the spacing-discharge relationships. The effect of (Y (or K(p) and p(O)] on the

TABLE I1 Parametric Soil Data

Gilat (loam) Mahal-Sinai (sand)

-40 -15

0.014 0.142

0.56 3.15

0.025 0.062

0.28 0.11

-80 -5 5

-40 4 0

5.15 X 2.35 x lo-'

0.367 0.166

TABLE 111 Midway Distances (rc) between Trickle Emitters Calculated by Linearized Steady State Solution (Lin. Soln.) and Numerical Transient State Solution (Num. Soh.) for Two Soils and Two Rates of Discharge Q Pu Pu rcVc) 'c(Pc) (Lin. Soln.) (Num. S o h ) a (Uter/ (Lin. Soh.) (Num. Soln.) W C ) Soil hour) (cm) (cm) (apu/2)' (Fig. 11)' (cm) (cm) Cilat (loam)

Nahal-Sinai (sand)

4 20

21.1 65.2

21.0 61.1

0.32 0.98

1.49 1.22

31.4 79.5

33.1 66.1

4

5.7 18.9

5.9 17.9

0.18 0.59

3.55 2.10

20.2 39.7

20.7 30.3

20

aDimensionless.

TRICKLE-DRIP IRRIGATION 250

0

I

-

4

I I p c = -70 cm

0

DISCHARGE

12

Q

385

I

16

20

liter

(-) hour

FIG. 13. Distance between emitters ( d ) as a function of trickle discharge Q) for two soils and two values of pC After Bresler (1977).

spacing-distance (d-Q) relationships for a given p c is illustrated in Fig. 13. According to Philip (1968), a is a measure of the relative importance of gravity and capillary for water movement in a particular soil. In soil with a high a value gravity tends to dominate and in soil with low (Y values capillary tends to dominate. This tendency is more pronounced at smaller rates of discharge (Q). Figure 13 also illustrates the effects of soil-water properties and the rate of discharge on the selection of spacing between emitters. Larger spacing is permitted in soils with lower values of K , and a (see Table I1 and Fig. 13) and also when the crop grown is not sensitive to water stresses and/or to partial soil wetting (higher p c values are permitted). Closer spacing is required for soils having higher K , and 01 values when a sensitive crop is being grown. For any given soil the emitter spacing can be increased as the discharge rate becomes higher. However, since the rate of growth of rc or d with Q decreases as Q increases (Figs. 12 and 13), the proper choice of Q chiefly depends on some optimization criteria in the engineering design of the field irrigation system.

F. DESIGNING THE LATERAL SYSTEM

The data as given in Fig. 12 or 13 can be combined with principles of hydraulics in order to obtain diameter (0) and length ( L ) of the lateral system for design purposes (Bresler, 1977). For a given d-Q combination and uniformity criteria, the L-D design relationship and the necessary pressure head at the lateral inlet depend upon emitter discharge function, elevation changes, reduc-

386

ESHEL BRESLER

tion coefficient for dividing flow, and pipe roughness coefficient. The HazenWilliams equation accounting for dividing flow between emitters is

HL = 2.78.

F*L*D-4*8'(6N/C)1.85

(33)

where HL = friction head loss in laterals (meters) = average rate of emitter discharge (meters3 hours-') N = number of emitters per lateral F = reduction coefficient for dividing flow between emitters along the lateral L = the lateral length (meters) D = inside diameter of the lateral pipe (meters) C = the Hazen-Williams roughness coefficient Equation (33) is an empirical equation. Care should be taken in using the units specified to each of the above-dimensioned variables. The empirical values of F and C have been tabulated (compare, e.g., Howell and Hiler, 1974). As the spacing between emitters along the lateral is given by d = 2r, (Figs. 12 and 13), it is possible to express N as

=4

N = -L (34) 2r, d where d is the distance between emitters along the lateral. Substituting L / d for N in Eq. (33) and rearranging L = 88.88 D'.708 (HL/F)0.351(Cd/a)"649

(35)

Knowing the Q-d relationship (Fig. 12 or 13) and assuming F to be constant over a given range of L and d, Eq. ( 3 5 ) gives the L-D relationships for any preselected value of head loss HL . To select an appropriate value of HL for Eq. ( 3 9 , a proper criterion may be based on the differences between the emitter discharge at the lateral inlet and the downstream discharge, relative to the average discharge, Qi-Qd

GE

Q Here Qi is the inlet discharge, Qd is the downstream discharge, and E is a preselected error fraction, say 0.05 or similar. The relationship between emitter discharge rate and the hydraulic head at the emitter may be given by the empirical expression Q=b@

(37)

where b and p are constant characteristic of the flow regime in the emitter and H is the hydraulic head at the emitter. Data of b and are available from the

TRICKLE-DRIP IRRIGATION

387

manufacturer but also can easily be determined experimentally in the laboratory. Using the maximum value of e it follows from Eq. (36), using Eq. (37), that

H f = -€0+Pd b

Since HL

(38)

= Hi - H d , then

Knowing both the pressure head at the lateral inlet and the emitter constants b and 0,permits the calculation of HL from Eq. (39) for a given and Q. This value of HL is then substituted into Eq. (35) to obtain the D-L relationship needed for the lateral design. When the lateral length is given by the size of the plot and Hi is known, the diameter D is calculated from Eq. (35). Otherwise, the optimum economic 0-L-Hi combination has to be calculated for each field and soil condition. In summary, it is suggested (Bresler, 1977) that steady and nonsteady infdtration models, which are well suited for the analysis of unsaturated flow through porous media, can be applied to design a trickle irrigation system. The two modeling approaches make it possible to calculate the spacing between emitters as a function of their rate of discharge, soil hydraulic properties, and crop sensitivity to water stress. However, it is important to remember that each of the proposed methods has certain limitations. For example, some of them involve errors that arise from the linearization procedure and from the estimation of K , and a. The assumption concerning the steady state flow is a very restricting one. It should also be emphasized that problems are involved in selecting the correct hydraulic parameters of the soil: K(O) and p(O). In addition, seepage of unused water below the rooting zone is not considered when S/S, (Fig. 1I), O(r,o), or p(r,o) is taken at the soil surface. It is also emphasized that problems are involved in seiecting the correct p c value. Additional research is needed to ascertain the validity of the views expressed in this chapter, to develop field methods for determining the necessary soil-water parameters, and to select the best midspace pressure head for a given set of soil, climate, and crop growing conditions (Bresler, 1977).

VIII. Water Management in Marginal Soils

Hardpans of various types, different sands and sand dunes, desert pavement of many kinds, and saline and alkali soils are very common marginal soils in arid

388

ESHEL BRESLER

zones of the world (see, e.g., Gile, 1961; Litchfield and Mabbutt, 1962; Ives, 1959; Marbut, 1935; Burvill, 1956; Pennefather, 1951). In addition, in many areas of the humid tropics, soils are leached and become very acidic. Soil acidity is generally associated with aluminum toxicity, which limits the rooting depth, especially of those crops which are sensitive to aluminum (Charreau, 1974; Wolf and Drosdoff, 1974; Wolf, 1975). Most of the above-mentioned marginal soils are characterized by low values of “available water” and/or “water-holding capacity,” properties often associated with limited rooting systems. Moreover, in many parts of the humid tropics and of semiarid zones, rains are either inadequate in total amount or irregular in annual distribution. This unfavorable climate-soil combination tends to produce soil-water deficits which in turn cause water management to be a critical factor for successful agriculture. This is so because of the unfavorable soil-water properties of these marginal soils in combination with the occurrence of long dry periods and the limited root growth owing to hardpans, desert pavement, salinity, aLkalinity or acidity, and aluminum toxicity. Obviously, a crop yield can not be obtained without irrigation during the dry seasion, in any kind of soil. It appears that, since irrigation must be applied in these areas, a trickle irrigation system may be the preferred one in these marginal soils, for the following reasons. The method is capable of delivering water into the soil in small quantities as often as desired, so as to maximize irrigation frequency without any additional costs. As irrigation frequency increases, the infiltration period becomes the most important part of the irrigation cycle. When irrigation frequency is sufficiently high, so that the irrigation cycle is dominated by infiltration rather than by the extraction stage, the water-holding capacity or water-availability properties of any marginal soil become relatively unimportant. This is so because the soil-water regime is continuously maintained at a relatively high water-content level so that water is supplied to the crop as it is needed and there is no need to store water within the limited soil-root zone (Rawlins, 1973). Not having to bring water from storage to the limited root zone also eliminates the possible negative effect of fluctuations in soil-water content. The effect is probably more severe as the soil becomes more marginal with respect to its water-holding properties. Water management under high-frequency trickle irrigation therefore renders the unfavorable water-storage properties of many marginal soils essentially unimportant. Irrigation management therefore involves optimization in the design of spacing between emitters and the lateral system (Section VII), as well as control of the quantities of water to be applied in order to meet the crop requirements and to supply the amount of water needed t o pass through the effective root zone to avoid salinity buildup (see Section VI). The quantity of water to be applied by an emitter in each single irrigation for any day after planting may be calculated from

TRICKLE-DRIP IRRIGATION

389

4 '(t)= R '(r)&(t)I(t)m$

(40)

where 4' is the quantity of water to be applied by an emitter at the day t after irrigation, E, is the class A pan evaporation between the preceding and the present irrigation, Z is the irrigation interval, r , is the radius of area wetted by an emitter, and R'(t) = ET(t)/E,(t) is the ratio between evapotranspiration (ET) and E, for the period t. This ratio as a function of number of days after irrigation, must be obtained experimentally. Note that this calculation does not take into account the quantity that must pass the effective root zone to avoid the hazard of salinity, which was discussed in Section VI. An additional management problem common to many marginal soils is that they may have a low fertility status-as in sands or sand dunes, or a high capacity to fuc phosphorus so as to make it unavailable to crops [e.g., marginal soils in the humid tropics (Wolf, 1975)l. The combination of low fertility status, high aluminum concentration, and phosphorus fixation may create an additional serious soil limitation to agricultural development. These soil fertility problems may be controlled by applying fertilizers simultaneously with irrigation through the trickling system (Shani, 1973). By using the proper management practice, one is able to optimize this system with respect to the nutritional balance and water status. LIST OF SYMBOLS The following symbols are used in this chapter: a = ap,/2 = parameter proportional to the length scale, dimensionless b = constant, Lz T-' c = solute concentration in the soil solution, C = Hazen-Williams roughness coefficient, dimensionless C, = solute concentration of the irrigation water, I W L - ~ D = inside diameter of the lateral pipe, L

d = distance between emitters, L = soil diffusion coefficient, L 2 T-I = hydrodynamic dispersion tensor, L 2 T-' E = evaporation flux, LT-' E , = class A pan evaporation rate, LT-' ET = evapotranspiration rate, LT-' F = reduction coefficient for dividing flow, dimensionless (? = average vertical pressure head gradient over the ponded area at the soil surface, dim ensionless H = hydraulic head, L Hi = inlet head, L Hd = downstream head, L HL = head loss in lateral, L I = irrigation interval, T

D, Dq

390

ESHEL BRESLER

K$ = saturated hydraulic conductivity tensor, LT-' K,.(e) = relative hydraulic conductivity, dimensionless K = K ( 0 ) = K @ ) = Kr(e)KS= capillary conductivity in isotropic media, LT-' L = lateral length, L N = number of emitters per lateral, dimensionless p = pore-water pressure head, L pa = air entry value of p , L Q = rate of discharge from emitter, L 3 T-' Qi = inlet discharge, L 3 T-' Q d = downstream discharge, L 3 T ' q = specific solution flux (Darcy's velocity), LT-' q' = quantity of water to be applied by an emitter, L 3 r = radial coordinate, L R = radial boundary of flow region, L R' = ratio between evapotransipration and class A pan evaporation, dimensionless rw = radius of the wetted area, L S = S(0) = S@) = transform water content, transform pore-water pressure head, L z T-' r = time, T T = end time of infiltration, T x i = Cartesian coordinate, L x, = vertical coordinate, L x,y = horizontal coordinates, L Y = crop yield per unit land area, V = average solution flow velocity, LT-' z = vertical coordinate, L Z = vertical boundary of flow region, L 01 = constant, L-' p = constant, dimensionless y = horizontal or radial coordinate, L f = relative vertical coordinate, dimensionless 0 = volumetric water content, dimensionless @ = soil-water regime index, dimensionless 5 = average value of 0,dimensionless @ = deviation of 0 from &, dimensionless E = discharge difference fraction, dimensionless uz = variance of @(t), dimensionless h~ = longitudinal dispersivity, L AT = transversal dispersivity, L 5 = relative radial coordinate, dimensionless p = radius of the ponded-water entry zone, L Subscrzp rs

o = reference value usually air-dry water content N =

initial value

s = value at saturation u = ultimate value, limiting value c = selected critical midway value

TRICKLE-DRIP IRRIGATION

39 1

REFERENCES Ayers, A. D., Wadleigh, C. H., and Magistad, C. C. 1943. J. A m . SOC.Agron. 35, 796-810. Bear, J. 1972. “Dynamics of Fluids in Porous Media,” pp. 5 7 9 6 6 2 . Am. Elsevier, New York. Bernstein, L., and Francois, L. E. 1973. Soil Sci. 115, 73-86. Bernstein, L., and Francois, L. E. 1975. Agron. J. 67, 185-190. Black, J. D. F., and West, D. W. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 4 3 2 4 3 6 . Boaz, M. 1973. “Trickle Irrigation in Israel.” Isr. Minist. Agric., Ext. Serv., Tel Aviv. Braester, C. 1973. Water Resour. Res. 9,687-694. Brandt, A., Bresler, E., Diner, N., Ben-Asher, I., Heller, J., and Goldberg, D. 1971. Soil Sci. SOC.Am., Proc. 3 5 , 6 7 5 4 8 2 . Bresler, E. 1973. Water Resour. Res. 9, 975-986. Bresler, E. 1975. Soil Sci SOC.Am., Proc. 3 9 , 6 0 4 4 13. Bresler, E. 1977. Irrigation Sci. 1 (in press). Bresler, E., and Russo, D. 1975. Soil Sci. SOC.A m . , Proc. 39, 585-586. Bresler, E., and Yaron, D. 1972. Water Resour. Res. 8, 791-800. Bresler, E., Heller, J., Diner, N., Ben-Asher, I., Brandt, A., and Goldberg, D. 1971. Soil Sci. Soc. Amer., Proc. 3 5 , 6 8 3 4 8 9 . Burvill, G. H. 1956. J. Dep. Agric., West. Aust., Salt Land Sum. 5 , 113-1 19. Charreau, C. 1974. “Soils of Tropical Dry and Dry-Wet Climatic Areas of West Africa and their Use and Management,” Agron. Mimeo. 74-26. Dep. Agron., Cornell Univ., Ithaca, New York. Childs, S. W., and Hanks, R. J. 1975. Soil Sci. SOC.Am., Proc. 39, 617-622. Christiansen, J. E. 1942. C a l q , Agric. Exp. Sm., Bull. 670. Dan, H. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 491-496. Dasberg, S., and Steinhardt, R. 1974. In: “Isotopes and Radiation Techniques in Soil Physics and Irrigation Studies. Proc. Series: 4 6 7 4 7 4 . I.A.E.A., Vienna. Frith, G. J. T., and Nichols, D. G. 1974. Proc. In?. Drip. Irrig. Congr., 2nd pp. 4 3 4 4 3 6 . Gardner, W. R. 1958. Soil Sci. 85, 228-232. Gerard, C. J. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 329-331. Gile, L. H. 1961. Soil Sci. SOC.A m . , Proc. 25, 5 2 4 1 . Goldberg, D., and Shmueli, M. 1970. Trans. Am. SOC.Agric. Eng. 13, 38-41. Goldberg, S. D., Rinot, M., and Karu, N. 1971. Soil Sci. SOC.Am., Proc. 35,127-130. Gornat, B., Goldberg, D., Rimon, D., and Ben-Asher, J. 1973. J. Am. SOC. Hortic. Sci. 98(2), 202-205. Grobbelaar, H. L., and Lourens, F. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 41 1-415. GUStdfSon, C. D., Marsh, A. W., Branson, R. L., and Davis, S. 1974. Proc. Inr. Drip Irrig. Congr., 2nd pp. 17-22. Haise, H. R., and Hagen, R. M. 1967. In “Irrigation of Agricultural Land” (R. M. Hagen, H. R. Haise, and T. W. Edminster, eds.), Agronomy, Vol. 11, pp. 577-597. A.S.A. Publ., Madison, Wisconsin. Halevy, I., Boaz, M., Zohar, Y., Shani, M., and Dan, H. 1973. In “Trickle Irrigation,” F A 0 Irrig. Drainage Pap. No. 14, pp. 75-1 17. F A 0 UN, Rome. Hanks, R. J., Klute, A., and Bresler, E. 1969. Water Resour. Res. 5, 1064-1069. Hart, W. E. 1961. Agric. Eng. 42(7), 354-355. Heller, J., and Bresler, E. 1973. In “Arid Zone Irrigation” (B. Yaron, E. Danfors, and Y. Vaadia, eds,), pp. 339-351. Springer-Verlag, Berlin and New York. Hiler, E. A., and Howell, T. A. 1973. Trans. A m . SOC.Agric. Eng. 16(4), 799-803.

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Hillel, D. 1972. In “Optimizing the Soil Physical Environment Toward Greater Crop Yields” (D. Hillel, ed.), pp. 79-100. Academic Press, New York. Howell, T. A., and Hiler, E. A. 1974. Trans. Am. Soc. Agric. Eng. 17,902-908. Isob, M 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 4 0 5 4 0 8 . Ives, R. L. 1959. A m . J. Sci 257(6), 4 4 9 4 5 7 . Kameli, D. 1971. Proc, Znt. Drip Irrig. Congr., l s t , Tel Aviv pp. 1-15. Kemper, W. D., and Noonan, L 1970. Soil Sci Soc. Am., Proc. 34, 126-130. Lange, A., Aljibury, F., and Fischer, B. 1974. Proc. Znt. Drip Irrig. Congr., 2nd pp. 422-424. Lemon, E. R. 1956. SoilSci. Soc. Am., Proc. 20,120-125. Lemos, P., and Lutz, J. F. 1957. Soil Sci Soc. Am., Proc. 21,485-491. Lindsey, K . E., and New, L. 1974. Proc. Znt. Drip Zrrig. Congr., 2nd pp. 40ft404. Litchfield, W. H., and Mabbutt, J. A. 1962. J. Soil Sci. 13, 148-159. Lomen, D. O., and Warrick, A. W. 1974. Soil Sci. Soc. A m . , Proc. 38, 568-576. Lunin, J., and Gallatin, M. H. 1965. Soil Sci. Soc. Am., Proc. 29,608-612. McElhoe, B. A., and Hilton, H. W. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 215-220. Marbut, C. F. 1935. In “Atlas of American Agriculture,” Vol. 3, Advanced Sheet No. 8. U.S. Dep. Agric., Washington, D.C. Neuman, S. P. 1973.J. Hydrol. Div., Proc, A m . Sac. CivilEng. 99(HYl2), 2233-2250. Ogata, A. 1970. US. Geol. Sum, Prof. Pap. 411-1. Olsen, S. R., and Kemper, W. D. 1968. Adv. Agron. 20, 81-151. Patterson, T. C., and Wierenga, P. J. 1974. Proc. Int. Drip. Irrig. Congr., 2nd pp. 376-381. Peleg, D., Lahav, N., and Goldberg, D. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 203-208. Pennefather, R. R. 1951. West. Mail 6 6 , 5 9 6 1 . Perkins, T. K., and Johnston, 0. C. 1963. SOC.Pet. Eng. J. 3, 70-84. Philip, J. R. 1968. Water Resour. Res. 4(5), 1039-1047. Philip, J. R. 1971. Soil Sci Soc. Am., Proc. 35, 867-871. Raats, P. A. C. 1971. Soil Sci Soc. Am., Proc. 35,689-694. Raats, P. A. C. 1972. Soil Sci. Sac. Am.. Proc. 36, 399-401. Rawitz, E. 1970. Soil Sci. 110131, 172-182. Rawlins, S. L. 1973. Soil Sci. Soc. Am., Proc. 37(4), 626-629. Rawlins, S. L. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 209-211. Rawlins, S. L., and Raats, P. A. C. 1975. Science 18,604-610. Rawlins, S. L., Hoffman, G. J., and Merrill, S. D. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 184-1 87. Rolland, L. 1973. In “Trickle Irrigation,” F A 0 Irrig. Drainage Pap. No. 14, pp. 3-73. F A 0 UN, Rome. Rose, C. W. 1961. Soil Sci. 9 1 , 4 9 4 4 . Safran, B., and Panes, B. 1975. “Nitrogen Fertilization Trials in Trickle Irrigated Vineyards.” Isr. Minist. Agric., Ext. Serv., Rehovot. (In Hebrew.) Seginer, I. 1967. Agric. Meteorol. 4, 281-291. Seginer, I. 1969. J. Irrig. Drainage Div., Proc. Am. Soc. Civil Eng. 95(IR2), 261-274. Shalhevet, J., and Bernstein, L. 1968. Soil Sci. 106, 85-93. Shani, M. 1973. “Techniques for Coupling Fertilization and Irrigation.” Isr. Minist. Agric., Ext. Serv., Rehovot. (In Hebrew.) Talsma, T. 1963. Meded. Landbouwhogesch. Wageningen 63(10), 1-68. Taylor, S. A. 1952. Soil Sci. 74,217-226. Tranter, C. J. 1951. ‘‘Integral Transformations in Mathematical Physics.” Methuen, London. Tscheschke, P., Alfaro, J. F., Keller, J., and Hanks, R. J. 1974. Soil Sci. 117,226-231. Wadleigh, C. H., and Ayers, A. D. 1945. Plant Physiol. 20, 106-132.

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Wadleigh, C. H., Gandi, H. G., and Kolisch, M. 1951. SoilSci. 72, 275-282. Warrick, A. W. 1974. SoilSci. SOC.Am., Proc. 39,383-386. Warrick, A. W., and Lomen, D. 0. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 228-233. Warrick, A. W., and Lomen, D. 0. 1976. Soil Sci. Soc. Am., J. 41,639-643. Waterfield, A. E. 1973. In “Trickle Irrigation,” F A 0 Irrig. Drainage Pap. No. 14, pp. 147-153. F A 0 UN, Rome. Wilke, 0. C. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 188-192. Willens, A. F., and Willens, G. A. 1974. Proc. Int. Drip Irrig, Congr., 2nd pp. 388-393. Willoughby, P., and Cockroft, B. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 439-445. Wolf, J. M. 1975. Ph.D. Thesis, Cornell Univ., Ithaca, New York. Wolf, .I. M., and Drosdoff, M. 1974. “Soil-water Studies on Oxisols and Ultisols of Puerto Rico,” Agron. Mimeo. 74-22. Dep. Agron. Agric. Eng., Cornell Univ., Ithaca, New York. Wooding, R. A. 1968. Water Resour. Res. 4(6), 1259-1273. Yagev, E., and Choresh, Y. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 456-461. Yaron, B., Shalhevet, J., and Shimshi, D. 1973. I n “Physical Aspects of Soil Water and Salts in Ecosystems” (A. Hadas, D. Swartzendruber, P. E. Rijtema, M. Fuchs, and B. Yarou, eds.), pp. 389-394. Springer-Verlag, Berlin and New York. Zaslavsky, D. 1972. In “Optimizing the Soil Physical Environment Toward Greater Crop Yields” (D. Hillel, ed.), pp. 223-232. Academic Press, New York. Zaslavsky, D., and Mokady, R. S. 1966. Soil Sci. 104, 1-6. Zentmyer, G. A., Guillemet, F. G., and Johnson, E. L. V. 1974. Proc. Int. Drip Irrig. Congr., 2nd pp. 512-514.

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SUBJECT INDEX A

white, 301 yellow, 301, 305-306 Brach iaria brachylopa, 7 mutica, 7, 8 ru&osa, 7 Brassica campestris, 45 napus, 57 oleracea, 4 3 Bromegrass, 57, 72 Bromus inermis, 57 tetorum, 131 Brown leaf spot, 217-278 narrow, 278 Buckwheat, 92 Bulbostylis aphylanthoides, 7

Agrobacterium tumefaciens, 66-69, 72 Aikiochi, 277 Alfalfa,43,60, 120, 121, 122, 124,127, 128,130,133 environment and growth, 183-227 Algae, blue-green, 9 AlIophane, physical properties, 229-264 Aluminum, 205,206 Ammonia, 29, 145, 147, 164 Ammophyla arenaria, 1 3 Andosol, 230 Andropogen gayanus, 7 SPP., 7 , 8 Asclepias syrica, 162, 163 Asparagus, 57 Asparagus offcinalis, 5 7 Astragalus cicer, 133 Atrazine, 162, 172-175 Azotobacter, 12 chroococcum, 1 3 , 2 2 paspali, 5 , 1 2 , 14-15,28, 32 spirillum, 15 vinelandii, 13, 14,20, 21, 72

C Cajanus cajan, 8 Calcium, 205, 206, 216 Capsicum annuum, 62 frutescens, 348 Carrot, 42, 51, 57, 58, 72, 73 Cassave, 23,43 Cell culture, genetic manipulation, 39-81 Cercospora oryzae, 278 Cheatgrass, 131 Chernozem, 107 Chilo suppressalis, 301 Chiseling, 143, 147-150,155-156, 163, 173,177 Citrus sinensis, 57 Clostridium, 1 2 pasteurianum, 2 1 Clover, 93,94, 216 alsike, 124, 130, 133 crimson, 124, 130 cup, 123 red, 124, 127, 130, 133 strawberry, 133 subterranean, 97,98, 121, 122, 124, 133 white, 98, 130, 133 Cochliobolus miyabeanus, 277 Cocksfoot, 217

B Bacillus maceranus, 11 megatherium var. phosphaticum, 108 polimyxa. 1 1 , 2 1 sp., 32 Bacterial blight, 280-286, 322, 323 leaf streak, 287 streak, 287 Barley, 4 3 , 4 5 , 5 8 , 6 3 , 126, 129 Bean, 119 Beijerinckia, 6, 10, 13-14, 22 fluminensis, 14 indica, 14 Black shank, 47 Blast disease, 267-275, 322, 323 Borer, pink, 301 striped, 301-305

395

396

SUBJECT INDEX

Coffee, 43 Corn, 92,108, 125,126, 216 see also maize borer, European, 169 disease control, 169 leaf blight, southern, 52 tillage-planting systems and yield, 141-182

Flax, 51 Flooding-tolerance, 54 Flowering, 212 Foxtail, 131 Fulvic acid, 85, 86

Coronilla varia, 133 Corticum saskii, 215 Cowpea, 51 Crepis, 44 Crown gall, 61 rust, 331 Cucumber, 126 Cucumis sativus, 126 Culture, anther and haploids, 44-48 cell, 40-44 Cynoden dactilon, I Cyperus obtusiflorus, I rotundus, I SP., 7

Gall midge, 318-322 Gene manipulation, 65-13 Genetics, bacterial blight resistance, 282-286 blast resistance, 265-215 disease resistance, 276-217,218, 219, 281,295,291 environment adaptation, 211-219 response, 185-1 86 insect resistance, 303-305, 309-313, 316-318,320-322 multiple resistance breeding, 322-333 somatic cell, 39-81 tungeo resistance, 292-294 Germination, 121-123,213 Gibbsite, 234 Glycine max, 5 1 Grass, nitrogen fixation, 1-38 salt marsh, 10 Grassy stunt, 294-296, 322, 323 Growth, leaf, 188-189 legume seedling, 119-1 39 root, 189-191 shoot, mathematical model, 186- .181

D Dactylis glomerata, 211 Dacus carota, 51 Dahlia pinnata, 12 Derxia gummosa, 14 Diabrotica longicornis, 169 Digitaria decumbens, nitrogen fixation, 2,5, 6, I , 26,29,30 Diplanthera wrightii, 12 Disease control, 169 resistance, 41, 52-53, 265-341 Disking, 145,141, 153,163, 114 Dormancy, 184,209 E Energy requirements, tillage-planting systems, 169-180 Enterobacter cloacae, 9, 10, 11, 12

F Fern, bracken, 23 Fertilization, 350, 313 Fertilizer, placement, 163-1 65

G

H Haplopappus. 44 Helminthosporium maydis, 5 2 oryzae, 211 Herbicides, 144, 146, 111 Hoja blanca, 299-301 Hordeurn bulbosurn, 61 jubaturn, 131 culgare, 6 1 Humic acid, 85,86 Humin, 85 Humus, 86,103 Hy parrh enia dissolu ta, I mfa, 7, 8

397

SUBJECT INDEX I

M

Imogolite, 230 Inositol phosphate, 86-88,91,93, 103, 107 Insect control, 169 resistance, rice, 301-322 Irrigation, 210 trickle-drip, 343-393

Maize, 43,49,51,53,54, 71, 107 see also corn nitrogen fixation, 8-9, 13,19,23,24,26, 27, 29, 30,31 Manganese, 20,205,206 Mangrove, 12 Manure, 101, 102, 111 Medicago asiatica, 186 falcata, 185, 203,213 glutinosa, 185 hispida, 129 lupulina, 203 sativa, 133,185, 186, 203,206 Melilotus alba, 122,133 officinalis, 133 Melinis rninuliflora, 7 Mentek, 288 Methodology, cell protoplasts, 55-60 haploid culture, 44-46, 47 mutant isolation, 48-55 organic phosphorus analysis, 84 soil dispersing, 232-234 Milkweed, 162,163 Millet, 108 Mineral, interrelationship, 207-209 root growth,204 uptake, 204-206 Mineralization, 98,100-112 Moisture, mineralization, 103-104 Mold, blue, 47 Montmorillonite, 2 38 Mycorrhizae, 94-95,108

J Juncus balticus, 12

K KlebsieNa aerobacter, 12 pneurnoniae, 72 Kresek. 280

L Laodelphax striatellus, 3 15 Lasso, 172-176 Leaf blight, corn, 169 Leafhopper, green, 292, 316-318, 322,327, 330 green rice, 298, 318 zigzag, 298 Legume, 5 1 seedling growth, 119-1 39 Lespedeza, 124 cuneata, 133 sripufacea, 133 striata, 133 Leucena glauca, 8 Light, growth, alfalfa, 191-195 Lilium, 44 Lime, 205 Linum usitatissirnurn, 57 Lolium, 189 perenne, 92, 98 rigidum, 98 LOIOX,173-176 Lotus cornicutatus, 133 purshianus, 129 Lucerne, 13 Lycopersicon escutenturn, 5 7

N Nephotettix cincticeps, 298, 315, 318 virescens, 288, 316 Nicotiana, 45,47, 62 glauca, 58, 59 glu tinosa, 47 langsdorfi, 5 8,59 tabacurn, 41,57,59 Nilaparvata lugens, 294,307 Nitrapyrin, 164 Nitrogen, 85,105,106, 164, 216 nitrate, 128, 349

398

SUBJECT INDEX

Nitrogen fixation, 206-207 efficiency, 21-22 gene manipulation, 71-73 grass, 1-38 Nodulation, 127, 128, 206, 207 No-till, 146,147,149-150,153,155,156, 163,169,175-176,177

0 Oat, 43, 108,215 Onobrychis viciifolia, 133 Orange, 57 Orange leaf virus, 288 Oryza nivara, 295, 327 Oxygen, nitrogen fixation, 17-18,28-29 P Pachydriplosis oryzae, 318 Panicum diehotomiflorum, 163 maximum, 7, 8,23,24,25,27 Panicum, fall, 163 Paraquat, 175-176 Paspalum comersenii, 7 notatum, nitrogen fixation, 2,5, 7, 14-15,27,28,30,32 Pasture, 97-98 Pea, 57, 119 Pennisetum americanum, 25 purpureum, 7, 29 Pepper, bell, 348 red, 62 Peronospora tabacina, 47 Phaseolus spp., 119, 126,193 Photomorphogenesis, 192 Photoperiod, 187, 191-192 Photosynthesis, 3,189, 194 Phosphabacterium, 108 Phosphorus, 129,147,164,204,216 grassland cycle, 96-97 inorganic, 93, 97, 105-106 soil organic, 83-1 17 Physiological predetermination, 120-1 21 Phytophthora, 203, 204 megasperma, 203 nico tianae, 4 7 Pinus radiata, 94 Pisum sativa, 57 spp., 119

Plant association, 214-217 Plant hopper, 299 brown, 307-313,323,323,330 small brown, 296, 315 white-backed, 314 Plowing, 130-131, 143,148-150, 153, 155,156,166,172-173,177 Podzol, 85,97 Potamogeton filiformis, 12 Potassium, 128,147, 164, 207, 208 Potato, 43 Protoplast, plant cell, 55-61 Pseudomonas tabaci, 5 2 Pyricularia oryzae, 267

R Rape, 12 Rapeseed, 57 Recilia dorsalis, 298 Rhizobium, 24, 32, 72, 131 Rhizoctnoica solani, 275 Rhizophora mangle, 12 Rhodospirillum rubrum, 20, 21 Rice,43,45,46,47,51,72 blast, 267-275, 322, 323 cadang cadang, 288 delphacid, 314-315 disease and insect resistance, 265-34 1 dwarf, 288,298 nitrogen fixation, 9-10, 13, 27 Ridge system, 145, 149, 150,155, 159, 167,174,177 Root rot, 351 Rootwork, Northern, 169 Ryegrass, 9

S Saccharum sp., 57 Sainfoin, 120,121,122,123,125,126, 128.133 Salinity, 348, 352 Salt-tolerance, 5 3-54 Sacrification, 122 Seedbed preparation, 130-131 Seedling growth, legume, 119-139 Sesarnia inferens, 301 Sheath blight, 275-27 7 Sogatella furcifera, 3 14, 3 15

399

SUBJECT INDEX Sogatodes cubanus, 299 oryzicola, 299, 314, 315, 322 Soil, allophane, 229-264 compaction, 159-162,254-256 erosion prevention, 166-169 solution, phosphorus, 90-95 stabilization, 260 thermal conductivity, 238-241 volcanic ash, 229 Soil-water management, trickle-drip irrigation, 343-393 Solanurn, 45 Sorghum, 7 1 nitrogen fixation, 8-9, 19, 26, 27, 29, 30 Soybean, 43, 57,58,60,92, 108 tillage-planting systems and yield, 141-182 Spartina alternijlora, 12 Spirillurn lipoferurn, 6, 8 , 10, 11, 12, 13, 15-26,27,28,31, 32,71 ecological distribution, 22-24 physiology, 17-22 Spirodela oligorrhiza, 94, 108 Stem borer, 301-306 Stem-rust, 331 Stress-resistance, 5 3-55 Stunt disease, 288 Strip disease, 296-298 Stylosanther harnata, 43 Sugar cane, 6, 13,43,57 Sulfur, 85, 105,204, 207 Superphosphate, 97 Sweet clover, 130 white, 122, 124, 133 yellow, 124,133 Sweet potato, 23 Syringodiurn filiforrne, 12

Tobacco, 45,46,47,51, 57,64,72 Tomato, 13,43,55,57,63 Transformation, plant, 61-73 Trefoil, big, 130 birdsfoot, 120, 122, 127, 130, 133, 137 narrow leaf, 124 Trerna cannabina, 72 Trifluralin, 163 Trifolium cherileri, 123 fragiferurn, 133 hirturn, 129 hybridurn, 133 incarnaturn, 129 rnicrocephalurn, 129 protense, 133 repens, 98,133 subterraneum, 97,98,120, 128, 133 tridentaturn, 129 Triticale, 45 Tryporyza incerrulas, 301, 305 innotata, 301 Tungro, 288-294,322 Turnip rape, 45

v Vetch, cicer milk, 121, 128,133 common, 133 crown, 120, 121,133, 137 woolypod, 123 Vicia dasycarpa, 123 sativa, 133 Vigna unguiculata, 57 Virus, tobacco mosaic, 47

W T Temperature, 127-128 growth effect, 195-199, 213 legume germination, 122-1 23 mineralization, 102-103 tillage systems and soil, 158-159 Thalassia testudinurn, 12 Till-planting, 146, 149-150, 167, 175, 177 Tillage-planting systems, yield and energy requirement, 141-182

Water conservation, 165-166 flow, modeling, 353-367 growth effect, 199, 200-204 trickle-drip irrigation, 343-393 retention, 247-250 transmission, 250-252 uptake, 199-200 Weed control, 162-163 Wheat, 43,45,47,51,12, 107, 108 nitrogen fixation, 10-11,13, 23, 27, 32 Wildfire disease, 52

SUBJECT INDEX

X Xanthomonas oryzae, 280 transtycens orizicola, 281

Yield, improving soil-water regime, 346-348 Z

Y

Zostera marina, 12

Yellow dwarf virus, 288 Yellow-orange leaf, 288

A

7

6 8 c 9 D O

E

l

F 2 G 3

H 4 1 5

1 6